Optical Scanning System

ABSTRACT

A scanning sensor system  100,  methods and kits for use thereof including a switchable light source  102,  a detector  106,  a substrate  104  and a plurality of optical sensing sites  112  are provided. Substrate  104  is coupled to and in optical communication with switchable light source  102  and detector  106.  Additionally, substrate  104  includes a plurality of substantially parallel excitation waveguides  108,  and a plurality of substantially parallel collection waveguides  110,  the excitation waveguides  108  and collection waveguides  110  crossing to form a two-dimensional array and optical communication with intersection regions  114  at each crossing. The plurality of optical sensing sites  112  are each in optical communication with an intersection region  114.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.60/743,458, filed Mar. 10, 2006 entitled Optical Scanning System, whichis incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Biological substance analysis methods based on optical means have risenin popularity in the last couple of decades. Common to all these methodsis that chemical interactions between the bio-molecules produce changesthat affect some measurable optical properties, such as emissionspectrum, absorption spectrum and index of refraction. The changes inthe optical properties can occur in the analyte itself or through amediator such as the surface on which the interaction takes place. Thesechanges are then monitored using a beam of incoming light (usually laserlight) which in-turn changes the outgoing light spectrum (e.g. influorescence), intensity (e.g. in absorption), or phase (e.g. SurfacePlasmon Resonance=SPR and any kind of interferometric method).

While most of these optical bio-analysis methods have found nicheapplications and markets, one method that became highly popular andinfluential was microarray optical fluorescence scanning. Such opticalscanning has enabled running tests on tens of thousands of miniaturesamples in a relatively short period of time. The major advantages ofthis method include: a) performance (sensitivity & Signal to NoiseRatio=SNR); b) speed; and c) miniaturization of the sampled analyte.These parameters define the efficiency and superiority of the method.

Currently microarray elements are spotted on top of a flat substratechip usually made of glass, plastic or epoxy. Subsequently, the chip isscanned using confocal scanning systems where the exciting light and theresulting fluorescence light are both shined and collected from aboveand analyzed using a single photo-multiplier (PMT) detector. Thisarrangement suffers from several inherent limitations including a veryshort interaction length between the bio-sample and the light (usually asingle mono-layer). This limits the signal strength and thus the SNR.Another limitation is a high background or noise due to the fact thatthe back reflected light and the emitted fluorescent light travel in thesame direction. A further limitation is high sensitivity to theplanarity and the position of the chip that need to be maintained infocus. Still another limitation is slow operation due to the need tohave large enough number of ‘pixels’ (scanned spots) within every sampleand long enough integration time. Yet another limitation is the need fora complicated optical and mechanical structure that entails bulky andexpensive systems.

Another optical bio-analysis method is waveguide based bio-sensors.Bio-sensing based on waveguides has been around for a while. Thesebiosensors can be divided into three main categories. The first involveslab waveguide fluorescence excitation with light collection from aboveor below the chip. In this arrangement the bio-analyzed spots arelocated on the surface of a chip that contains a single slab-waveguide.Light is coupled into the waveguide using a lens or a grating thatexcites the entire chip with all its bio-analyzed spots simultaneously.The fluorescence is collected using an optical imaging system and aCharge-Coupled Device (CCD) detector from above or underneath the chip.One drawback of this kind of systems is relatively poor performance dueto uniformity of excitation as well as collection of the light. Thisleads to non-repeatable results. Another drawback is high noise levelsdue to crosstalk between the different spots. A further drawback is thatlarge spots and relatively small numbers of elements are required togenerate signal large enough for efficient imaging with the CCD. Yetanother drawback is the long integration time to overcome SNR issues.Examples of the above method are described in U.S. Pat. Nos. 5,814,565;6,911,344 and 6,395,558.

A second waveguide based bio-sensor utilizes an interferometric opticaldevice. In this case, channel waveguides are used together withinterferometric devices such as Mach Zehender Interferometers (MZI) orring-resonators. These sensitive interferometric devices sense thechange in the index of refraction due to binding of the bio-moleculesnear a waveguide surface. The major problems associated with this typeof systems include non-specificity due to inability to recognize theexact reason for the index change which may occur from other materialdeposition as well as temperature changes. Another problem is a veryslow speed in addressing the different elements which disqualify thismethod for running large numbers of element arrays. Examples of theabove method are described in U.S. Pat. Nos. 5,494,798, 4,515,430,5,623,561 and 6,618,536.

A third waveguide based bio-sensor utilizes Surface Plasmon Resonance(SPR). Here, in one example, a thin gold layer deposited on top of aglass substrate. The bio-analyzed sample on top of the gold induceschanges in the refractive index above the gold layer and thus changingthe resonant angle for generating surface Plasmons along the gold layer.The Plasmons generation is detected as an enhanced peak in the reflectedbeam. Examples of the SPR method are covered, for example, in U.S. Pat.No. 6,956,651 B2. Other types of optical bio-sensors and array scannersexist such as U.S. Pat. No. 6,396,995 B1.

SUMMARY OF THE INVENTION

In general, in one aspect, the invention features a scanning sensorsystem, methods and kits for use thereof including a switchable lightsource, a detector, a substrate and a plurality of optical sensingsites. The substrate is coupled to and in optical communication with theswitchable light source and the detector. Additionally, the substrateincludes a plurality of substantially parallel excitation waveguides,and a plurality of substantially parallel collection waveguides, theexcitation waveguides and collection waveguides crossing to form atwo-dimensional array and optical communication with intersectionregions. The plurality of optical sensing sites are each in opticalcommunication with an intersection region.

Implementations of the invention can include one or more of thefollowing features.

In one aspect of the invention a scanning sensor system for detecting abiologically active analyte is provided. The system includes aswitchable light source, a detector and a substrate coupled to and inoptical communication with the switchable light source and the detector.The substrate includes a plurality of substantially parallel excitationwaveguides, and a plurality of substantially parallel collectionwaveguides, the excitation waveguides and collection waveguides crossingto form a two-dimensional array of intersection regions where anexcitation waveguide and a collection waveguide cross and provideoptical communication with the intersection region at each crossing. Thesystem further includes a plurality of optical sensing sites each inoptical communication with an intersection region.

In one embodiment, the system is substantially planar. In a particularembodiment the system includes a planar lightwave circuit.

In another embodiment the switchable light source is coupled to and incommunication with one or more of the excitation waveguides at a firstedge of the substrate and the detector is coupled to and incommunication with one or more of the collection waveguides at a secondedge of the substrate.

In one embodiment the optical sensing sites include a sensor including abiologically active analyte in a sample, and wherein a measurable changein the first light wave results when the sensor discriminates orinteracts with the biologically active analyte.

In one embodiment of the system a first light wave generated by theswitchable light source in an excitation waveguide is transduced by asensor of an optical sensing site in optical communication with theexcitation wave guide resulting in a second light wave in a collectionwaveguide, the second light wave being detectable by the detector.

In another embodiment of the system the sensor is adapted to support animmunoassay. In a particular embodiment the immunoassay supported is anenzyme-linked immunosorbent assay (ELISA). In another embodiment theimmunoassay supported is a fluorescent immunoassay. In yet anotherembodiment the sensor is selected from a fluorescence well, anabsorption cell, an interferometric sensor, a diffractive sensor and asurface plasmon resonance sensor.

In one embodiment of the system the biologically active analyte isselected from a nucleic acid, a protein, an antigen, an antibody, amicroorganism, a gas, a chemical agent and a pollutant. In a particularembodiment the nucleic acid is produced via an amplification reaction.

In one embodiment of the system the excitation waveguides aresingle-mode and the collection waveguides are multi-mode. In anotherembodiment the excitation waveguides support single-mode in a firstvertical dimension and multi-mode in a second lateral dimension andwherein the collection waveguides are multi-mode. In a furtherembodiment the excitation waveguides and the collection waveguides aremulti-mode. In another embodiment the excitation waveguides and thecollection waveguides are single-mode. In yet another embodiment theexcitation waveguide comprises a plurality of branches for drawing afraction of the light from a first light wave traveling in theexcitation waveguide. In a related embodiment the excitation waveguidebranches are in optical communication with the excitation waveguide.

In one embodiment of the system the collection waveguide include aplurality of funnels for collecting light from the sensing sites andcoupling it to the collection waveguide. In a particular embodiment theoptical sensing sites comprise wells. In one embodiment the opticalsensing sites include the surface of the substrate above theintersection region of the excitation waveguides and the collectionwaveguides. In another embodiment the optical sensing sites includebiochemical interaction sites. In a further embodiment the opticalsensing sites include optical transducers. In one embodiment the opticaltransducers include fluorescence wells comprising fluorescent orluminescent compounds, wherein light waves guided by the excitationwaveguides excite the fluorescent or luminescent compound in the wellsin the presence of a biologically active analyte, and the collectionwaveguides collect and guide light emitted from the wells to thedetector.

In one embodiment of the system the switchable light source includes adynamic light source. In one embodiment the switchable light sourceincludes a chip containing an array of light generators coupled to anarray of waveguides. In another embodiment the switchable light sourceis an optical switch including a light generator coupled to one or moreinput of the optical switch. In a particular embodiment the opticalswitch further includes a branched architecture. In one embodiment theoptical switch further includes one or more inputs and multiple outputs.In another embodiment the optical switch further includes greater thanabout 10 outputs. In yet another embodiment the optical switch furtherincludes greater than about 100 outputs. In a further embodiment theoptical switch further includes greater than about 1,000 outputs. In oneembodiment the optical switch further includes substantially between 50and 500 outputs. In another embodiment the switchable light source isbutt-coupled to the substrate. In yet another embodiment the switchablelight source includes one or more waveguide and is evanescently coupledto the substrate through a proximate arrangement of the one or moreswitchable light source waveguide and one or more excitation waveguideof the substrate.

In one embodiment of the system the light generator provides variablewavelengths of light. In another embodiment the light generator isselected from the group consisting of a broad-band source, a source withone or more discrete spectral lines and a tunable source.

In one embodiment of the system the detector is a photodetector array.In a particular embodiment the detector is a plurality of detectors. Inone embodiment two or more detectors are coupled to and in opticalcommunication with one or more of the collection waveguides or theexcitation waveguides at one or more edges of the substrate.

In one embodiment of the system the number of intersection regions isgreater than 10. In another embodiment the density of intersectionregions is greater than 100 per cm². In yet another embodiment thedensity of intersection regions is greater than 2,000 per cm².

One embodiment of the system further includes a thermal transfer elementin thermal communication with the substrate. In a particular embodimentthe thermal transfer element is a thermoelectric cooler. In oneembodiment each optical sensing site includes a thermal transfer elementin thermal communication with the optical sensing site. In anotherembodiment the thermal transfer element includes a thin-film heater. Inone embodiment each optical sensing site further includes a thermistorin thermal communication with the optical sensing site.

In one embodiment of the system the substrate further includes one ormore microchannel and one or more reservoirs in fluid communication withone or more optical sensing site. In one embodiment the system furtherincludes a fluidics layer coupled to the substrate and comprising one ormore microchannel and one or more reservoirs in fluid communication withone or more optical sensing site.

In another aspect the invention provides scanning sensing methodincluding delivering a sample suspected of containing a biologicallyactive analyte to be detected to an optical sensing site of a scanningsensor system, providing a first light wave using a switchable lightsource to one or more of a plurality of substantially parallelexcitation waveguides in optical communication with the optical sensingsite, wherein the first light wave is transducable by a sensorassociated with the optical sensing site to a second light wave carriedin one or more of a plurality of substantially parallel collectionwaveguides in optical communication with the optical sensing site andcrossing the excitation waveguides. The method further includesdetecting a measurable change in the second light wave using a detectorin optical communication with the collection waveguides, wherein ameasurable change in the first light waves occurs when the sensorinteracts with the biologically active analyte.

In one embodiment scanning sensing further includes switching one ormore input light wave from the switchable light source into thesubstrate to produce the first light wave in one or more of theexcitation waveguides. In a particular embodiment the switchable lightsource includes an optical switch for controlled switching of one ormore input light wave, the optical switch can multicast light to aplurality of outputs and into the substrate to controllably produce thefirst light wave in one or more of the excitation waveguides. In oneembodiment the switchable light source includes an array of individuallycontrolled light generators for controlled switching of one or moreinput light wave, to controllably produce the first light wave in one ormore of the excitation waveguides.

In one embodiment scanning sensing further includes simultaneouslydetecting the second light wave with the detector at the end of eachcollection waveguide wherein the detector comprises a photodetectorarray. In one embodiment a portion of the sensing sites includereference sample material for calibration and/or normalization.

In one embodiment the biologically active analyte is selected from anucleic acid, a protein, an antigen, an antibody, a microorganism, agas, a chemical agent and a pollutant. In a particular embodiment thebiologically active analyte is a protein. In one embodiment a SNP isdetected in the biologically active analyte. In another embodimentexpression of a gene is detected upon detection of the biologicallyactive analyte.

In one embodiment the sensor of the scanning sensing method is adaptedto support an immunoassay and the sensor interacting with thebiologically active analyte includes an outcome of an immunoassay. Inone embodiment the immunoassay supported is an enzyme-linkedimmunosorbent assay (ELISA). In another embodiment the immunoassaysupported is a fluorescent immunoassay.

In one embodiment of the method detecting a measurable change in thesecond lightwave provides a diagnostic result. In another embodiment themethod includes conducting a real-time PCR reaction at the opticalsensing site.

In one aspect the invention provides a kit for assaying a sample for abiologically active analyte including a scanning sensor systemcomprising a switchable light source, a detector, and a substratecoupled to and in optical communication with the switchable light sourceand the detector. The substrate includes a plurality of substantiallyparallel excitation waveguides, and a plurality of substantiallyparallel collection waveguides, the excitation waveguides and collectionwaveguides crossing to form a two-dimensional array of intersectionregions where an excitation waveguide and a collection waveguide crossand provide optical communication with the intersection regions at eachcrossing, and a plurality of optical sensing sites each in opticalcommunication with an intersection region. The kit also includespackaging and instructions for use of the system. In one embodiment thesystem includes a planar lightwave circuit. In another embodimentcrossings of the excitation waveguides and collection waveguides aresubstantially perpendicular. In a particular embodiment the opticalsensing sites include a sensor adapted to support an immunoassay, andthe kit further includes one or more immunoassay reagents.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

A better understanding of the features and advantages of the presentmethods and compositions may be obtained by reference to the followingdetailed description that sets forth illustrative embodiments, in whichthe principles of our methods, compositions, devices and apparatuses areutilized, and the accompanying drawings of which:

FIG. 1A is a schematic of the scanning sensing system of the inventionincluding a switchable light source, a substrate, a detector and opticalsensing sites.

FIG. 1B is block diagram showing the scanning system of the invention aspart of a working system in a housing.

FIG. 2A is a schematic of the substrate of the invention includingexcitation and collection optical waveguides in conjunction with opticalsensing sites and barriers.

FIG. 2B is a perspective view of the substrate including excitation andcollection optical waveguides in conjunction with optical sensing sites.

FIG. 2C is a schematic of two cross-sectional views (AA and BB) of thesubstrate shown in FIGS. 2A and 2B.

FIG. 2D is a schematic of a side view of the substrate in relation to athermoelectric cooler.

FIG. 2E is a schematic of the substrate of the invention illustratingdetails of an optical sensing site including a heater and a thermistor.

FIG. 2F is a schematic of the substrate of the invention includingreservoirs and micro channels in relation to optical sensing sites.

FIG. 3A is a schematic of the substrate of the invention includingexcitation and collection optical waveguides in conjunction with opticalsensing sites, barriers and funnels.

FIG. 3B is a schematic showing and enlarged view of substrate featuresshown in FIG. 3A.

FIG. 3C is a schematic of a cross-sectional view of the substrate shownin FIG. 3B.

FIG. 4A is a schematic of the substrate of the invention includingexcitation and collection optical waveguides in conjunction with opticalsensing sites, barriers and branches.

FIG. 4B is a schematic showing and enlarged view of substrate featuresshown in FIG. 4A.

FIG. 4C is a schematic of a cross-sectional view in a plane (AA) of thesubstrate shown in FIG. 4B.

FIG. 4D is a schematic of a cross-sectional view in a plane (BB) of thesubstrate shown in FIG. 4B.

FIG. 5A is a schematic of a general substrate including typical layersand waveguides representative of those of the current invention.

FIG. 5B is a photomicrograph image of waveguides representative of thoseof the invention and a silica layer.

FIG. 5C is a perspective view of waveguides and associated layers.

FIG. 6A is a schematic of the switchable light source of the inventionincluding inputs and outputs.

FIG. 6B is a schematic of a branched architecture between the inputs andoutputs of the switchable light source.

FIG. 6C is a schematic of another embodiment of the switchable lightsource of the invention including light generators and waveguides.

FIG. 7 is a schematic of a detector of the invention.

FIG. 8 is a block diagram showing a representative example logic devicein communication with an apparatus for use with the scanning sensingsystem of the invention.

FIG. 9 is a block diagram showing a representative example of a kit.

FIGS. 10A-D are schematics illustrating a representative manufacturingprocess for the substrate and waveguides of the invention.

FIG. 11 is a flow chart showing a representative manufacturing processfor the substrate.

DETAILED DESCRIPTION OF THE INVENTION

Apparatus, methods, and kits for optical sensing, using a scanningsensing system including a switchable light source, a detector asubstrate and a plurality of optical sensing sites are provided. Thesubstrate of the system includes a plurality of substantially parallelexcitation waveguides and a plurality of substantially parallelcollection waveguides. The excitation waveguides and collectionwaveguides cross to form an intersection region and a two-dimensionalarray. The optical sensing sites include a sensor and are in opticalcommunication with one or more excitation waveguides and one or morecollection waveguides. Sensing of a variety of environmental andbiological samples can be achieved using the apparatus, methods and kitsdescribed herein. The general theoretical principles of lightwaveguiding and evanescent field fluorescence excitation apply to theembodiments disclosed herein.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the inventions described herein belong. Although anymethods, devices, and materials similar or equivalent to those describedherein can be used in the practice or testing of the inventionsdescribed herein, the preferred methods, devices and materials are nowdescribed.

Definitions

The term “biologically active analyte” when used herein means anysubstance which can affect any physical or biochemical properties of abiological organism, including but not limited to viruses, bacteria,fungi, plants, animals, and humans. In particular as used herein,biologically active analyte according to the present invention includeswithout limitation drugs, prodrugs, pharmaceutical agents, drugmetabolites, biomarkers such as expressed proteins and cell markers,antibodies, serum proteins, cholesterol, polysaccharides, nucleic acids,biological analytes, gene, protein, or hormone, or any combinationthereof. A biologically active analyte can further include a natural orman-made substance including but not limited to a gas, a chemical agentor a pollutant, or a combination thereof (e.g., from an environmentalsource). At a molecular level, the biologically active analytes can bepolypeptide glycoprotein, polysaccharide, lipid, nucleic acid, and acombination thereof.

Of particular interest are biomarkers associated with a particulardisease or with a specific disease stage. Such biologically activeanalytes include but are not limited to those associated with autoimmunediseases, obesity, hypertension, diabetes, neuronal and/or musculardegenerative diseases, cardiac diseases, endocrine disorders, anycombinations thereof.

Also of interest are biomarkers that are present in varying abundance inone or more of the body tissues including heart, liver, prostate, lung,kidney, bone marrow, blood, skin, bladder, brain, muscles, nerves, andselected tissues that are affected by various disease, such as differenttypes of cancer (malignant or non-metastatic), autoimmune diseases,inflammatory or degenerative diseases.

Also of interest are biologically active analytes that are indicative ofa microorganism. Exemplary microorganisms include but are not limited tobacterium, virus, fungus and protozoa. Biologically active analytes thatcan be detected by the subject method also include blood-born pathogensselected from a non-limiting group that consists of Staphylococcusepidermidis, Escherichia coli, methicillin-resistant Staphylococcusaureus (MSRA), Staphylococcus aureus, Staphylococcus hominis,Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus capitis,Staphylococcus warneri, Klebsiella pneumoniae, Haemophilus influnzae,Staphylococcus simulans, Streptococcus pneumoniae and Candida albicans.

Biologically active analytes that can be detected by the subject deviceand methods also encompass a variety of sexually transmitted diseasesselected from the following: gonorrhea (Neisseria gorrhoeae), syphilis(Treponena pallidum), chlamydia (Chlamydia tracomitis), nongonococcalurethritis (Ureaplasma urealyticum), yeast infection (Candida albicans),chancroid (Haemophilus ducreyi), trichomoniasis (Trichomonas vaginalis),genital herpes (HSV type I & II), HIV I, HIV II and hepatitis A, B, C,G, as well as hepatitis caused by TTV.

Additional biologically active analytes that can be detected by thesubject apparatus and methods encompass a diversity of respiratorypathogens including but not limited to Pseudomonas aeruginosa,methicillin-resistant Staphylococcus aureus (MSRA), Klebsiellapneumoniae, Haemophilis influenzae, Staphylococcus aureus,Stenotrophomonas maltophilia, Haemophilis parainfluenzae, Escherichiacoli, Enterococcus faecalis, Serratia marcescens, Haemophilisparahaemolyticus, Enterococcus cloacae, Candida albicans, Moraxiellacatarrhalis, Streptococcus pneumoniae, Citrobacter freundii,Enterococcus faecium, Klebsiella oxytoca, Pseudomonas fluorsecens,Neisseria meningitidis, Streptococcus pyogenes, Pneumocystis carinii,Klebsiella pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae,and Mycobacterium tuberculosis.

Listed below are additional exemplary markers according to the presentinvention: Theophylline, CRP, CKMB, PSA, Myoglobin, CA125, Progesterone,TxB2, 6-keto-PGF-1-alpha, and Theophylline, Estradiol, Lutenizinghormone, High sensitivity CRP, Triglycerides, Tryptase, Low densitylipoprotein Cholesterol, High density lipoprotein Cholesterol,Cholesterol, IGFR.

Exemplary liver markers include without limitation LDH, (LD5), (ALT),Arginase 1 (liver type), Alpha-fetoprotein (AFP), Alkaline phosphatase,Alanine aminotransferase, Lactate dehydrogenase, and Bilirubin.

Exemplary kidney markers include without limitation TNFa Receptor,Cystatin C, Lipocalin-type urinary prostaglandin D, synthatase (LPGDS),Hepatocyte growth factor receptor, Polycystin 2, Polycystin 1,Fibrocystin, Uromodulin, Alanine, aminopeptidase,N-acetyl-B-D-glucosaminidase, Albumin, and Retinol-binding protein(RBP).

Exemplary heart markers include without limitation Troponin I (TnI),Troponin T (TnT), CK, CKMB, Myoglobin, Fatty acid binding protein(FABP), CRP, D-dimer, S-100 protein, BNP, NT-proBNP, PAPP-A,Myeloperoxidase (MPO), Glycogen phosphorylase isoenzyme BB (GPBB),Thrombin Activatable Fibrinolysis Inhibitor (TAFI), Fibrinogen, Ischemiamodified albumin (IMA), Cardiotrophin-1, and MLC-I (Myosin LightChain-I).

Exemplary pancreas markers include without limitation Amylase,Pancreatitis-Associated protein (PAP-1), and Regenerate in proteins(REG).

Exemplary muscle tissue markers include without limitation Myostatin.

Exemplary blood markers include without limitation Erythopoeitin (EPO).

Exemplary bone markers include without limitation, Cross-linkedN-telopeptides of bone type I collagen (NTx), Carboxyterminalcross-linking telopeptide of bone collagen, Lysyl-pyridinoline(deoxypyridinoline), Pyridinoline, Tartrate-resistant acid phosphatase,Procollagen type I C propeptide, Procollagen type I N propeptide,Osteocalcin (bone gla-protein), Alkaline phosphatase, Cathepsin K, COMP(Cartilage Oligomeric Matrix Protein), Osteocrin, Osteoprotegerin (OPG),RANKL, sRANK, TRAP 5 (TRACP 5), Osteoblast Specific Factor I (OSF-1,Pleiotrophin), Soluble cell adhesion molecules, sTfR, sCD4, sCD8, sCD44,and Osteoblast Specific Factor 2 (OSF-2, Periostin).

In some embodiments markers according to the present invention aredisease specific. Exemplary cancer markers include without limitationPSA (total prostate specific antigen), Creatinine, Prostatic acidphosphatase, PSA complexes, Prostrate-specific gene-1, CA 12-5,Carcinoembryonic Antigen (CEA), Alpha feto protein (AFP), hCG (Humanchorionic gonadotropin), Inhibin, CAA Ovarian C1824, CA 27.29, CA 15-3,CAA Breast C1924, Her-2, Pancreatic, CA 19-9, Carcinoembryonic Antigen,CAA pancreatic, Neuron-specific enolase, Angiostatin. DcR3 (Solubledecoy receptor 3), Endostatin, Ep-CAM (MK-1), Free Immunoglobulin LightChain Kappa, Free Immunoglobulin Light Chain Lambda, Herstatin,Chromogranin A, Adrenomedullin, Integrin, Epidermal growth factorreceptor, Epidermal growth factor receptor-Tyrosine kinase,Pro-adrenomedullin N-terminal 20 peptide, Vascular endothelial growthfactor, Vascular endothelial growth factor receptor, Stem cell factorreceptor, c-kit/KDR, KDR, and Midkine.

Exemplary infectious disease markers include without limitation Viremia,Bacteremia, Sepsis, PMN Elastase, PMN elastase/α1-PI complex, SurfactantProtein D (SP-D), HBVc antigen, HBVs antigen, Anti-HBVc, Anti-HIV,T-suppressor cell antigen, T-cell antigen ratio, T-helper cell antigen,Anti-HCV, Pyrogens, p24 antigen, Muramyl-dipeptide.

Exemplary diabetes markers include without limitation C-Peptide,Hemoglobin Alc, Glycated albumin, Advanced glycosylation end products(AGEs), 1,5-anhydroglucitol, Gastric Inhibitory Polypeptide, Glucose,Hemoglobin, ANGPTL3 and 4.

Exemplary inflammation markers include without limitation Rheumatoidfactor (RF), Antinuclear Antibody (ANA), C-reactive protein (CRP), ClaraCell Protein (Uteroglobin).

Exemplary allergy markers include without limitation Total IgE andSpecific IgE.

Exemplary autism markers include without limitation Ceruloplasmin,Metalothioneine, Zinc, Copper, B6, B12, Glutathione, Alkalinephosphatase, and Activation of apo-alkaline phosphatase.

Exemplary coagulation disorders markers include without limitationb-Thromboglobulin, Platelet factor 4, Von Willebrand factor.

In some embodiments a marker may be therapy specific. COX inhibitorsinclude without limitation TxB2 (Cox-1), 6-keto-PGF-1-alpha (Cox 2),11-Dehydro-TxB-1a (Cox-1).

Other markers of the present include without limitation Leptin, Leptinreceptor, and Procalcitonin, Brain S100 protein, Substance P,8-Iso-PGF-2a.

Exemplary geriatric markers include without limitation, Neuron-specificenolase, GFAP, and S100B.

Exemplary markers of nutritional status include without limitationPrealbumin, Albumin, Retinol-binding protein (RBP), Transferrin,Acylation-Stimulating Protein (ASP), Adiponectin, Agouti-Related Protein(AgRP), Angiopoietin-like Protein 4 (ANGPTL4, FIAF), C-peptide, AFABP(Adipocyte Fatty Acid Binding Protein, FABP4), Acylation-StimulatingProtein (ASP), EFABP (Epidermal Fatty Acid Binding Protein, FABP5),Glicentin, Glucagon, Glucagon-Like Peptide-1, Glucagon-Like Peptide-2,Ghrelin, Insulin, Leptin, Leptin Receptor, PYY, RELMs, Resistin, andsTfR (soluble Transferrin Receptor).

Exemplary markers of Lipid metabolism include without limitationApo-lipoproteins (several), Apo-A1, Apo-B, Apo-C-CII, Apo-D, Apo-E.

Exemplary coagulation status markers include without limitation FactorI: Fibrinogen, Factor II: Prothrombin, Factor III: Tissue factor, FactorIV: Calcium, Factor V: Proaccelerin, Factor VI, Factor VII:Proconvertin, Factor VIII:, Anti-hemolytic factor, Factor IX: Christmasfactor, Factor X: Stuart-Prower factor, Factor XI: Plasma thromboplastinantecedent, Factor XII: Hageman factor, Factor XIII: Fibrin-stabilizingfactor, Prekallikrein, High-molecular-weight kininogen, Protein C,Protein S, D-dimer, Tissue plasminogen activator, Plasminogen,a2-Antiplasmin, Plasminogen activator inhibitor 1 (PAI1).

Exemplary monoclonal antibody markers include those for EGFR, ErbB2, andIGF1R.

Exemplary tyrosine kinase inhibitor markers include without limitationAb1, Kit, PDGFR, Src, ErbB2, ErbB 4, EGFR, EphB, VEGFR1-4, PDGFRb, FLt3,FGFR, PKC, Met, Tie2, RAF, and TrkA.

Exemplary Serine/Threonine Kinase Inhibitor markers include withoutlimitation AKT, Aurora A/B/B, CDK, CDK (pan), CDK1-2, VEGFR2, PDGFRb,CDK4/6, MEK1-2, mTOR, and PKC-beta.

GPCR target markers include without limitation Histamine Receptors,Serotonin Receptors, Angiotensin Receptors, Adrenoreceptors, MuscarinicAcetylcholine Receptors, GnRH Receptors, Dopamine Receptors,Prostaglandin Receptors, and ADP Receptors.

For the purposes of this invention, a “therapeutic agent” is intended toinclude any substances that have therapeutic utility and/or potential.Such substances include but are not limited to biological or chemicalcompounds such as a simple or complex organic or inorganic molecules,peptides, proteins (e.g. antibodies) or a polynucleotides (e.g.anti-sense). A vast array of compounds can be synthesized, for example,polymers, such as polypeptides and polynucleotides, and syntheticorganic compounds based on various core structures, and these are alsoincluded in the term “therapeutic agent”. In addition, various naturalsources can provide compounds for screening, such as plant or animalextracts, and the like. It should be understood, although not alwaysexplicitly stated that the agent is used alone or in combination withanother agent, having the same or different biological activity as theagents identified by the inventive screen. The agents and methods alsoare intended to be combined with other therapies.

Pharmacodynamic (PD) parameters according to the present inventioninclude without limitation physical parameters such as temperature,heart rate/pulse, blood pressure, and respiratory rate, and biomarkerssuch as proteins, cells, and cell markers. Biomarkers could beindicative of disease or could be a result of the action of a drug.Pharmacokinetic (PK) parameters according to the present inventioninclude without limitation drug and drug metabolite concentration.Identifying and quantifying the PK parameters rapidly from a samplevolume is extremely desirable for proper safety and efficacy of drugs.If the drug and metabolite concentrations are outside a desired rangeand/or unexpected metabolites are generated due to an unexpectedreaction to the drug, immediate action may be necessary to ensure thesafety of the patient. Similarly, if any of the PD parameters falloutside the desired range during a treatment regime, immediate actionmay have to be taken as well.

In preferred embodiments physical parameter data is stored in orcompared to store profiles of physical parameter data in abioinformatics system which may be on an external device incorporatingpharmacogenomic and pharmacokinetic data into its models for thedetermination of toxicity and dosing. Not only does this generate datafor clinical trials years prior to current processes but also enablesthe elimination of current disparities between apparent efficacy andactual toxicity of drugs through real-time continuous monitoring. Duringthe go/no go decision process in clinical studies, large scalecomparative population studies can be conducted with the data stored onthe database. This compilation of data and real-time monitoring allowsmore patients to enter clinical trials in a safe fashion earlier thancurrently allowed. In another embodiment biomarkers discovered in humantissue studies can be targeted by the scanning sensing system forimproved accuracy in determining drug pathways and efficacy in cancerstudies.

The term “nucleic acid” when used herein refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The term “microorganism” when used herein refers to bacteria,actinomycetales, cyanobacteria (unicellular algae), fungi, protozoa,animal cells or plant cells or virus. Examples of microorganisms includebut are not limited to pathogens.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-natural amino acid. As used herein, the terms encompass amino acidchains of any length, including full length proteins (i.e., antigens),wherein the amino acid residues are linked by covalent peptide bonds. Inaddition, proteins that contain multiple polypeptide chains thatassociate through covalent and/or non-covalent interactions are alsoencompassed by “protein,” as used herein.

The term “polymorphism” as used herein refers to the occurrence of twoor more genetically determined alternative sequences or alleles in apopulation. A polymorphic marker or site is the locus at whichdivergence occurs. Preferred markers have at least two alleles, eachoccurring at frequency of greater than 1%, and more preferably greaterthan 10% or 20% of a selected population. A polymorphism may compriseone or more base changes, an insertion, a repeat, or a deletion. Apolymorphic locus may be as small as one base pair. Polymorphic markersinclude restriction fragment length polymorphisms, variable number oftandem repeats (VNTR's), hypervariable regions, minisatellites,dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats,simple sequence repeats, and insertion elements such as Alu. The firstidentified allelic form is arbitrarily designated as the reference formand other allelic forms are designated as alternative or variantalleles. The allelic form occurring most frequently in a selectedpopulation is sometimes referred to as the wildtype form. Diploidorganisms may be homozygous or heterozygous for allelic forms. Adiallelic polymorphism has two forms. A triallelic polymorphism hasthree forms.

A single nucleotide polymorphism (SNP) occurs at a polymorphic siteoccupied by a single nucleotide, which is the site of variation betweenallelic sequences. The site is usually preceded by and followed byhighly conserved sequences of the allele (e.g., sequences that vary inless than 1/100 or 1/1000 members of the populations).

A single nucleotide polymorphism usually arises due to substitution ofone nucleotide for another at the polymorphic site. A transition is thereplacement of one purine by another purine or one pyrimidine by anotherpyrimidine. A transversion is the replacement of a purine by apyrimidine or vice versa. Single nucleotide polymorphisms can also arisefrom a deletion of a nucleotide or an insertion of a nucleotide relativeto a reference allele.

The term “individual” when used herein is not limited to a human being,but may also include other organisms including but not limited tomammals, plants, bacteria or cells derived from any of the above.

Aspects of the invention may include one or more of the followingadvantageous features. Dense and accurate integration of opticalmanipulating elements can be achieved using planar lightwave circuitstechnology. Applications for planar lightwave circuits as describedherein include new drug discovery and development, disease research,biomarkers discovery, SNP association studies including toxicology anddisease susceptibility, and diagnostics including identifying patientspredisposed to diseases and identifying patients with particular drugsensitivity.

FIG. 1A illustrates an exemplary scanning sensing system 100 of theinvention including a switchable light source 102, a substrate 104,optical sensing sites 112 and a detector 106. The substrate includesexcitation waveguides 108 and collection waveguides 1 10 that cross orintersect at an intersection region 114.

In one embodiment, as shown in FIG. 1A, the switchable light source 102is coupled to and is in optical communication with one or more of theexcitation waveguides 108 at a first edge of the substrate 104.Additionally, the detector 106 is coupled to and in opticalcommunication with one or more of the collection waveguides 110 at asecond edge of the substrate 104. Although a single detector at one edgeof the substrate is shown, it is envisioned that two or more detectorscould be coupled to and in optical communication with one or morecollection waveguide at various edges of the substrate (not shown). Forexample, in one embodiment, where the switchable light source is coupledto a first edge of the substrate, a first detector could be coupled toan adjacent edge and be in optical communication with a first end of acollection waveguide, while a second detector could be coupled toanother adjacent edge and be in optical communication with a second endof a collection waveguide. A third detector can be coupled to the edgeopposite to the one coupled to the switchable light source and inoptical communication with the second end of the excitation waveguides(not shown).

As shown in FIG. 1A, in one embodiment the system 100 can besubstantially planar. For example, the switchable light source 102 canbe a planar chip. This can be coupled to a planar substrate 104 that isa second chip, that is further coupled to a planar detector 106 that isa third chip. In a particular embodiment, as shown in FIG. 1A, thesystem 100 is a planar lightwave circuit including three coupled chips.In one embodiment two chips are integrated into a single chip (e.g., anoptical switch chip and substrate chip). Such a configuration would beuseful in a case where the substrate chip is reusable and can beeffectively used for long periods of time. One application of such aconfiguration would be in a system for detecting biologicalwarfare-associated agents. In such an application it would beadvantageous for the system to operate for long periods of time withouta need for replacing the chip. In addition, having two chips integratedon a single substrate solves the problem of maintaining the relativealignment of two chips (e.g., a switchable light source chip andsubstrate chip).

Where the system is used in biological applications, including but notlimited to detection of biologically active analytes including nucleicacids, proteins or microorganisms, the substrate can be a multi-elementbio-analysis chip.

It is envisioned that crossing or intersecting of the excitationwaveguides and the collection waveguides can be a direct physicalcrossing or intersecting, for example, where the excitation waveguidesand the collection waveguides are embedded within the substrate in asingle or multiple layers. Alternatively, it is envisioned that thecrossing or intersecting involves a physical space or distance betweenthe excitation waveguides and the collection waveguides, for example,where the excitation waveguides and the collection waveguides areembedded within the substrate in separate layers. The optical sensingsites 112 of the system 100 typically are associated with theintersection regions 114. Typically one optical sensing site 112 isassociated with each intersection region 114. As illustrated, in oneembodiment the number of intersection regions 114 and optical sensingsites 112 is an arrangement of 100 intersection regions 114 and 100optical sensing sites 112. It is envisioned that the number ofintersection regions and optical sensing regions on a substrate chip canbe greater than 10, greater than 100, greater than 1,000 or greater than10,000. It is further envisioned that the density of intersectionregions can be greater than 10 per cm², greater than 100 per cm²,greater than 1,000 per cm² or greater than 10,000 per cm². In oneembodiment the density of intersection regions is greater than 2,000 percm².

As further shown in FIG. 1A, the crossing or intersecting of theexcitation waveguides 108 and the collection waveguides 110 can besubstantially perpendicular, for example, at an angle of 90°.Alternatively, in certain embodiments the crossing or intersecting canbe angled less than or great than 90°.

It is also envisioned that in any of the embodiments described herein,that a first light wave generated by the switchable light source in anexcitation waveguide induces the sensor to transduce an optical signalresulting in a second light wave in a collection waveguide, the secondlight wave being detectable by the detector.

As illustrated in FIG. 1A, in one advantageous embodiment, the system100 is a planar two-dimensional scanning system. The system 100 in thisembodiment includes a planar switchable light source 102, for example, aplanar optical switch or an array of switchable lasers, coupled to theplane of the substrate 104, for example, a bio-analysis chip plane.Furthermore, the switchable light source 102 can provide a dynamicsource of light for selective and programmed excitation in respect toindividual excitation waveguides 108, providing excitation to all of theoptical sensing sites 112 along that excitation waveguide 108. A dynamiclight source includes but is not limited to a tunable wavelength and/ortunable bandwidth light source. Additionally, the system 100 of thisembodiment provides for planar collection of the emitted light from allthe excited sensing sites 112 in the collection waveguides 110,specifically in the plane of the substrate 104, such that the lightcollection is substantially perpendicular to the direction of the lightproduced in the excitation waveguides 108.

FIG. 1B is an exemplary illustration of the scanning system of theinvention as part of a working system 101 in a housing 109. While thescanning sensing system illustrated in FIG. 1A is core of the presentinvention, in order to facilitate the operation of this system, one ormore other modules can be included in a working system that includes thescanning sensing system components of the invention.

FIG. 1B illustrates that working system 101 can include housing 109 forenclosing various modules of the working system including but notlimited to substrate 104, robotic system 103, switchable light source102, multi-element detector 106, electronic boards 107 and interfacepanel 105. Substrate 104, switchable light source 102, and multi-elementdetector 106, are discussed in detail below.

In regard to housing 109, as shown in FIG. 1B, in one embodiment anenclosure or housing 109 holds in place two fixed chips (e.g., of a3-chip architecture), namely, switchable light source 102 andmulti-element detector 106. Accordingly, in this embodiment substrate104 chip is movable in relation to switchable light source 102 andmulti-element detector 106. Housing 109 can include any number ofaccurately machined parts and or components and described herein,allowing, for example, the relative alignment of the 3 optical chips.The working system housing can optionally include temperature controland vibration isolation for the working system (not shown).

As shown in FIG. 1B, working system 101 can further include an X, Y, Z,θ robotic system 103 for positioning substrate 104 as required withinworking system 101. X, Y, Z, θ robotic system 103 can be a translationstage with several degrees of freedom for receiving or acceptingsubstrate 104, holding it in place, and aligning it in relation to therest of working system 101. As desired, at the end of a run X, Y, Z, θrobotic system 103 can eject substrate 104 from working system 101.

It is envisioned that the working system can further include an aligningsystem (not shown). An aligning system can include one or more lightsources, one or more detectors and one or more cameras for activedetection of the position of the substrate of the invention. Based onthe detected position, the aligning system can align the substrate tothe rest of the working system modules, for example, to provide alignedoptical communication between the substrate and the switchable lightsource.

As shown in FIG. 1B, working system 101 can further include one or moreelectronic boards 107, for example, an electronic driving board and acontrol board. It is envisioned that one or more electronic boards cancontrol all the different parts of the working system. Electronic boards107 can control switchable light source 102 and any other light sourcepresent in the system. Electronic boards 107 can be adapted to read anyor all of the detectors and cameras in the working system 101.Electronic boards 107 can further be adapted to drive robotic system 103and control its motion, and optionally monitor and control temperaturein different areas of the system. Electronic boards can include logicelements and processors (not shown). It is envisioned that electronicboards can further include embedded software both for controlling theworking system and for interfacing the outside world, for example by wayof the interface panel 105 having a key-pad or any other input/outputport.

As shown in FIG. 1B, working system 101 can additionally include one ormore interface panel 105. It is anticipated that the system will haveone or more interface panel 105 which allow a user to interface with thesystem and operate it. Interface panels can include any number of inputand output ports for connecting the system to other systems or to anexternal control console (not shown).

FIG. 2A illustrates an exemplary substrate 204 of the system of theinvention further including barriers 218 intended to block stray lightwithin the substrate and reduce crosstalk between the different elementsof the substrate. The barriers 218 can be light absorbing or lightreflecting. The barriers 218 can be variously sized, shaped andpositioned between the collection waveguides 210 and/or the excitationwaveguides 208 in any of a number of orientations to achieve a desiredoptical effect. As shown in FIG. 2A, the barriers 218 can be arranged ina row between two adjacent collection waveguides and proximal to theoptical sensing sites 212 and intersection region 214.

As shown in FIG. 2B, (in this view a top cladding layer is not shown) inone embodiment the substrate 204 can include excitation waveguides 208and collection waveguides 210 embedded beneath a surface of thesubstrate 204 in multiple layers. As shown, the excitation waveguides208 cross, physically intersect, and are in optical communication withthe collection waveguides 210 at the intersection regions 214. In theembodiment shown in FIG. 2B, the optical sensing site 212 is positionedat the intersection region 214 above and in optical communication withthe excitation waveguides 208. As further shown in FIG. 2B, thesubstrate 204 includes multiple layers including a Silicon layer 220 anda Silica (SiO2) layer 222, wherein the collection waveguides 210 areembedded within the Silica (SiO2) layer 222.

As shown in FIG. 2C, in another embodiment the substrate can includeexcitation waveguides 208 and collection waveguides 210 embeddedunderneath a surface of the substrate 204 in a single layer. As shown,the excitation waveguides 208 cross, physically intersect and are inoptical communication with the collection waveguides 210. In contrast tothe embodiment shown in FIG. 2B, here the intersection betweenexcitation waveguides 208 and collection waveguides 210 occurs internalto the collection waveguides 210. As further shown in FIG. 2C, thesubstrate 204 includes multiple layers including a Silicon layer 220, aSilica (SiO2) layer 222, and a cladding layer 224. As shown, theexcitation waveguides 208 and collection waveguides 210 can be embeddedwithin the Silica (SiO2) layer 222. Additionally, the optical sensingsite 212 can be embedded within both the cladding layer 224 and theSilica (SiO2) layer 222. Optionally, the optical sensing site can beembedded solely within the cladding layer (not shown).

It is envisioned that the excitation waveguides and collection waveguides can be single-mode or multi-mode waveguides. In one embodiment,the excitation waveguides are single-mode and the collection waveguidesare multi-mode. It is envisioned that waveguide configurations caninclude single- or multi-mode configurations in either vertical orlateral orientations within a waveguide. For example, in one specificand non-limiting embodiment, the excitation waveguides 208 can support asingle mode in the vertical dimension and multi modes in the lateraldimension. Optionally, as shown in FIG. 2A, the excitation waveguides208 and the collection waveguides 210 can span the entire substrate fromone edge to another edge.

As shown in FIG. 2C, the substrate 204 components and optical sensingsites 212 can include dimensions. FIG. 2C shows two cross-section viewsof the substrate 204. View AA is a cross-section view in plane A asindicated in FIG. 2A and FIG. 2B. View BB is a cross-section view inplan B as indicated in FIG. 2A and FIG. 2B. As shown in FIG. 2C, thethickness of the cladding layer 224 above the excitation waveguides canbe about 0.1 μm to 20 μm. In one embodiment the cladding layer 224thickness is about 1-2 μm. By way of a non-limiting example, as shown inFIG. 2C, an opening of the optical sensing site 212 can include thefollowing dimensions: about 20 μm×2 μm. The distance between collectionwaveguides 210 can range from about 1 μm to 1000 μm. For example, asshown in FIG. 2C, the distance between collection waveguides 210 can beabout 100 μm. The distance between collection waveguides 210 and theSilicon layer 220 can be about 1 μm to 100 μm. For example, as shown inFIG. 2C, the distance between collection waveguides 210 and the siliconlayer 220 can be about 10-20 μm.

As shown in FIGS. 2B and 2C, the excitation waveguides 208 andcollection waveguides 210 can be channel waveguides. Exemplary rangesfor waveguide dimensions in the embodiment shown in FIGS. 2B and 2Cinclude about 0.2 to 100 μm thick and about 1 to 100 μm wide. By way ofnon-limiting example only, the excitation waveguides 208 can includecross-section dimensions of about 0.5 μm×2 μm and the collectionwaveguides 210 can include cross-section dimensions of about 20 μm×20μm.

FIG. 2D in a side view illustrates another embodiment of substrate 204of the invention in relation to a thermal transfer element 203, forexample, a thermoelectric cooler (TEC). Thermal transfer element 203 isa temperature control system useful for heating or cooling a chip, forexample, substrate 204. Although the thermal transfer element may bereferred to herein as a cooling element, it is to be understood thatwhere the thermal transfer element is configured to increase anddecrease the temperature of a chip, the component functions essentiallyas a heating and as a cooling element depending on the induced directionof the electrical current. The thermal transfer element can provide arange of useful temperatures. For example, the thermal transfer elementcan be configured to provide a temperature in the range between about−40° C. to about 120° C. as desired. The thermal transfer 203 elementcan be adapted to receive substrate 204 of the invention. The thermaltransfer element 203 can be adapted to contact part or all of a surfaceof the substrate 204 of the invention.

Providing thermal transfer element 203 in conjunction with substrate 204of the invention is useful, for example, for the amplification of testedsample molecules through processes such as Polymerase Chain Reaction(PCR) as described herein. In use, the embodiment as described for FIG.2D provides the capability of controlling the temperature of the entiresubstrate such that as the temperature of the entire substrate iscycled, samples at any optical sensing site can be amplified by PCRsimultaneously.

FIG. 2E illustrates another embodiment of substrate 204 of the inventionwherein optical sensing site 212 includes heater 205 and thermistor 207.In this embodiment, optical sensing site 212 of substrate 204 caninclude heater 205, for example, a thin-film heater, in the vicinity ofeach sensing sites 212. Heater 205 can be adapted to enable individualtemperature control for each sensing site 212. In addition to heater205, thermistor 207 can be located at or near each sensing site 212thereby providing for measuring the local temperature. In use, thisembodiment provides the capability of running the same or any desireddifferent number of cycles and the same or any desired differenttemperature profiles for each and every sensing site.

Advantageously, the embodiments described for FIGS. 2D and 2E cansupport real-time PCR. As described herein, since optical detection isdone from within the substrate, signal detection in both embodiments(see FIGS. 2D and 2E) can be done while the samples are in the processof the amplification cycles, thereby enabling real time analysis of thePCR process.

FIG. 2F illustrates yet another embodiment of substrate 204 of theinvention wherein substrate 204 additionally includes reservoirs 211 andmicrochannels 209 in relation to optical sensing sites 212. As such, inthis embodiment microfluidics are incorporated into the substrate.Microfluidics can be adapted to drive liquid (in this case the testedsample) using the capillary effect across the substrate. As illustratedin FIG. 2F, this can be achieved by an arrangement of microchannels 209,optionally of varying width, which force the sample from one or morereservoirs 211 to optical sensing sites 212 which can include etchedwells to receive the sample. The microchannels can be either etched onthe face of the chip itself or can be added as an external structure ona surface of the sensing chip.

In use, it is envisioned that a sample to be tested can be pipetted intoa reservoir at one end of the substrate. The sample can then bedistributed using the microfluidic system to the optical sensing sitesand sensing wells where it is allowed to bind to pre-spotted probes andcan subsequently be optically scanned and analyzed. Several reservoirsmay be used to separate different samples/patients or for runningseveral parallel tests.

FIG. 3A in a top view illustrates an exemplary substrate 304 of thesystem of the invention wherein the collection waveguides 310 includefunnels 317 (shown in detail in FIG. 3B) for collecting light.

As shown in the example in FIG. 3A, the substrate 304 can include a10×10 array consisting of 10 excitation waveguides 308 (e.g., 5 μmwide×2 μm deep), 10 collection waveguides 110 (e.g., 30 μm wide×10 μmdeep), 100 optical sensing sites 312 (e.g., wells 30 μm long×5 μmwide×10 μm deep), 100 funnels 317 for collecting light from the opticalsensing sites 312 and barriers 318 (e.g., light absorbing channels) toreduce crosstalk between the optical sensing sites 312. Although theexample shown in FIG. 3A includes a 10×10 array of excitation waveguides308 and collection waveguides 110, it is envisioned that the substratecan include greater than 10, greater than 100 or greater than 1,000excitation waveguides 308 and collection waveguides 110.

In the embodiment shown in FIG. 3A, excitation light can be coupled intoone or more excitation waveguides 308 on the left hand side of thesubstrate 304 through, for example, chip-to-chip butt coupling.Excitation light can travel along the excitation waveguides 308 andcouple into the optical sensing sites (e.g., wells) through anevanescent field tail. Additionally, the switchable light source caninclude one or more waveguide and can be evanescently coupled to thesubstrate through a proximate arrangement of the one or more switchablelight source waveguide and one or more excitation waveguide of thesubstrate. Excited fluorescence generated in the optical sensing site312 can be collected along the long facet of the optical sensing site312 into the funnels 317. The funnels 317 can channel the light into thecollection waveguides 310. The light in the collection waveguides 310can be coupled out at the “bottom” of the substrate 304 into a detectorarray (not shown). Light scattered outside the optical sensing sites 312can be blocked by a series of barriers 318 (e.g., light absorbers) toavoid crosstalk between parallel collection waveguides 310.

In one embodiment, the substrate shown in FIG. 3A includes two waveguidelayers. As illustrated in cross-sectional view in FIG. 3C, a first 2 μmthick bottom layer can include the excitation waveguide 308. The bottomlayer can have a higher refractive index in order to increase theevanescent field tail presence in the optical sensing sites. An upper 10μm thick layer can contain the optical sensing site and the lightcollection structures (funnels and waveguides). The upper layer can havea lower refractive index than the bottom layer in order to minimizelight loss when coupling the light out of the substrate to the detector.

In a particular embodiment of the above, both the excitation andcollection waveguides are multimode. Furthermore, the switchable lightsource (e.g., an optical switch or an array of light generators coupledto an array of waveguides) can include single-mode waveguides, that canbe butt-coupled or can evanescently couple to the substrate.

As shown in cross-sectional view in FIG. 3C, in order to minimize theloss of light at the waveguide crossing points due to light couplingfrom the collection waveguides 310 into the excitation waveguides 308,the excitation waveguides 308 can be thinner than the collectionwaveguides 310. For example, as shown in FIGS. 3B and 3C, the excitationwaveguides 308 can have a width of 5 μm (see FIG. 3B) and a height of 2μm (see FIG. 3C). As further shown, the collection waveguides 310 canhave a width of 30 μm (see FIGS. 3A and 3B) and a height of 10 μm (seeFIG. 3C).

It is envisioned that light coupled at the waveguides crossing pointsbetween the excitation waveguides and the collection waveguides canshine directly into the optical sensing sites, thereby increasing lightexcitation rather than being lost.

As shown in FIG. 3B, the optical sensing sites can be wells that arenarrow (5 μm) and long (30 μm) with light collectable along the longfacet. Such a configuration increases the efficiency of lightcollection. In addition, light excitation coupling into the well canincreases due to the long coupling length. The well dimensions (5×30×10μm³) yield a volume of 1.5 pico-liter. Larger wells are also envisionedin a variety of sizes yielding volumes ranging from about 0.1 pico-literto 100 micro-liter.

The funnel can have a radii for the collection, confinement and couplingof light into the collection waveguides. Suitable ranges for radii caninclude from about 100 μm to about 1000 μm.

The barriers 318 as illustrated in FIGS. 3A and 3B, can be trenchesfilled with light absorbing material (e.g., a metal such as gold). Wherethe barriers 318 are trenches, the trenches can include openings abovethe excitation waveguide 308 to avoid loss at the crossing points (notshown).

The overall dimensions of the substrate illustrated in FIG. 3A can be1.2×1.2 mm². Margins can optionally be included around the substrate toadjust the overall dimensions as desired.

FIG. 4A illustrates an exemplary substrate 404 of the system of theinvention wherein the Excitation waveguides 408 include a plurality ofbranches 421 (shown in detail in FIG. 4B) for tapping light from theexcitation waveguides and coupling it into the sensing wells.

In the embodiment shown in FIG. 4A, the substrate 404 can be made up ofseveral waveguide layers (e.g., three waveguide layers). Such aconfiguration can be useful, for example, to optimize excitation andfluorescence collection while minimizing loss and crosstalk. FIGS. 4Cand 4D are schematic cross-section views of the substrate 404 throughplanes at (AA) and (BB) respectively as indicated in FIG. 4B.

In one embodiment the substrate consists of three waveguide layershaving core refractive index of 1.7 and clad reflective index of 1.4.Useful core refractive index values range from about 1.45 to 1.7, anduseful clad refractive index values range from about 1.4 to 1.44.

As shown in FIGS. 4C and 4D, in one embodiment where the substrate 404includes three waveguide layers, a first bottom layer can be about 10 μmthick and include the collection waveguides 410. In the embodimentillustrated in FIG. 4A, the collection waveguides 410 can be 30 μm wide,multimode and traverse the substrate 410 from substantially edge toedge. A second middle waveguide layer can be 0.5 μm to 1 μm thick andinclude coupling waveguide branches 421 (see FIGS. 4A and 4B). Thebranches 421 can couple excited light into the optical sensing sites,which can be wells. A third top layer can be 2 μm thick and includesingle-mode excitation waveguides 408 and traverse the substratesubstantially from edge to edge.

The substrate of the scanning sensing system can made up of any of anumber of well known materials suitable for use in planar lightcircuits. For example, useful substrate materials include but are notlimited to Silica (SiO2), glass, epoxy, lithium niobate and indiumphosphide as well as combinations thereof. The waveguides disclosedherein can be made up of Silicon, Silica (SiO2) and derivatives thereof,silicon oxynitride (SiON) and derivatives thereof, silicon nitride (SiN)and derivatives thereof, polymers, lithium niobate and indium phosphideas well as combinations thereof. In one embodiment, UV light is used tochange the refractive index of a waveguide material after deposition.

FIG. 5A illustrates an exemplary silicon layer 520 of the substrate 504.For example, the silicon layer 520 can be made up of a silicon waferhaving a thickness from about 0.1 mm to 10 mm. In another example thesilicon wafer can have a thickness from about 0.3 to 1 mm. In aparticular example as illustrated in FIG. 5A, the silicon wafer has athickness of 0.65 mm. As shown in FIG. 5A in one embodiment, the silica(SiO2) layer 522 is a 14 μm thermal oxide layer of Silica (SiO2) createdby placing the Silicon in an oxygen-rich environment inside a furnace athigh temperature. The top Silicon layer oxidizes over time (severalhours) creating a SiO2 layer. Additionally, as shown in FIG. 5A, in oneembodiment, the cladding layer 524 is 15 μm thick and deposited by aPECVD (Plasma-Enhanced Chemical Vapor Deposition) process after etchingto produce the waveguides 508.

It is envisioned that the various layers of the substrate can includedifferent refraction index properties. For example, a wafer layer ofsilicon has a higher refraction index than a cladding layer of silicadeposited thereon.

As shown in FIG. 5B (illustrated with a photomicrograph prior todeposition of a cladding layer), in some embodiments, the substrate 504can include two waveguides 507 arranged for light wave coupling on asilica (SiO2) layer 522. Alternatively, as shown in FIG. 5C, twowaveguides 508 can be arranged for guiding uncoupled light waves on asilica (SiO2) layer 522 and overclad with a cladding layer 524.

The optical sensing sites in one embodiment are in the form of wells,for example, etched wells (see FIG. 4D expanded view). Where the opticalsensing site is a well, it can act as a vessel for a liquid sample. Inanother embodiment the optical sensing sites are a region on the surfaceof the substrate, for example, above the intersection region of theexcitation waveguides and the collection waveguides (not shown). In afurther embodiment, the optical sensing sites are biochemicalinteraction sites. For example, where the optical sensing site is a wellcontaining a sensor single stranded DNA oligonucleotide having afluorescent tag attached, a solution containing a target complementarysingle stranded DNA added to the well could biochemically interact bybase-pairing with the sensor within the optical sensing site (notshown). In another example the optical sensing site is a location orwell containing one or more immunoassay reagent for conducting animmunoassay as described herein.

In a particular embodiment, the optical sensing sites comprise opticaltransducers (not shown). An optical transducer is defined as any devicethat generates a measurable change (wavelength, amplitude or phase) tothe incoming first light wave and can thus be monitored in the outgoingsecond light wave. In one embodiment the optical transducers arefluorescence wells including fluorescent or luminescent compounds,wherein light waves guided by the excitation waveguides excite thefluorescent or luminescent compound in the wells in the presence of atarget, and the collection waveguides collect and guide light emittedfrom the wells to the detector, for example at the edge of the chip (notshown).

The sensor of the optical sensing site of the system can be a sensorthat discriminates or interacts with a target (e.g., a biologicallyactive analyte) in a sample from, for example, a biological, man-made orenvironmental source. As discussed above, a first lightwave can inducethe sensor to transduce an optical signal to a second light wave. In oneembodiment where the sensor is capable of discriminating or interactingwith a target in a sample, a measurable change in the second light wavecan result when the sensor discriminates or interacts with the target.Upon detection of the change in the second light wave using the detectorof the system, presence of the target in the sample is known.

Any of a number of sensors can be used with the scanning sensing systemto measure phenomenon associated with sensing of a target in a sample.Examples of suitable sensors include, but are not limited to, afluorescence well or cell, an absorption cell, an interferometricsensor, a diffractive sensor or a surface plasmon resonance (SPR)detector. For a fluorescence well or cell, the phenomenon measurable canbe light emission from luminescent or fluorescent molecular tags. Forexample, emitted light at an altered wavelength can be measured. In thecase of an absorption cell, changes in the sample optical density (OD)can measurably affect the intensity of the light passing through thesample. For an interferometric sensor, changes in the effectiverefractive index of a waveguide generate a phase different between twobeams of light leading to different interference patterns measurable asa difference in intensity at the detector. For a diffractive sensor,changes in the effective refractive index at the surface of adiffractive element, for example, a grating, affect the diffractionangle of the light for a given wavelength or alternatively affect thewavelength at a given diffraction angle. In the case of a SPR sensor,changes in the effective refractive index at a metal-dielectricinterface affect the resonance conditions for generating surfaceplasmons.

FIG. 6A illustrates an exemplary switchable light source 602 of thesystem of the invention, including one or more inputs 601 as a primarysource of light for coupling to a light generator The light generatorcan be any source of electromagnetic radiation emitting one or morediscrete spectral-lines or a continuous spectrum (not shown). In onepreferred embodiment the light generator is a laser source emitting inone or more well defined wavelengths. In a second preferred embodimentthe light generator is a tunable laser that can be tuned to emit lightin one wavelength within a predefined range. As illustrated, switchablelight source 602 further includes a plurality of outputs 603 shown inFIG. 6A as N—Outputs. The number of outputs 603 included in switchablelight source 602 can be variable based on the intended use. For example,in certain applications the number of outputs 603 can be greater than 10outputs. In one embodiment the number of outputs 603 can be great than100 outputs. In a further embodiment the number of outputs 603 can begreater than 1,000 outputs. In another embodiment the number of outputs603 ranges from about 50 to 500. In a particular embodiment, the numberof outputs 603 is about 128. As shown in FIG. 6A, in one embodiment, theswitchable light source includes outputs 603 that fan out from an input601. As illustrated in FIG. 6B, in one embodiment a branchedarchitecture stemming from the input 601 to the outputs 603 can be used.Although only one input is shown in FIGS. 6A and 6B, it is envisionedthat multiple inputs 601 can be used.

FIG. 6C illustrates another exemplary switchable light source 602 of thesystem of the invention including multiple outputs. In this embodimentswitchable light source 602 includes a plurality of waveguides 609,light generators 607 and electronic leads 605. As shown in FIG. 6Cwaveguides 603 can be arranged in parallel across substrate 611. Inother embodiments waveguides are arranged in a non-parallel fashion (notshown). Waveguides 609 can terminate in outputs 603 as described herein.

As shown in FIG. 6C, light generators 607 as described herein can bearranged in optical communication with waveguides 609. As further shownin FIG. 6C, light generators 607 can be in electrical communication withelectronic leads 605. Electronic leads can in turn be in electricalcommunication with any of a number of apparatus including but notlimited to a power supply or an electronic driving circuit (not shown).

It is envisioned that the switchable light source can be a dynamic lightsource allowing for selective and programmed excitation through one ormore individual output. In one embodiment the switchable light source isan optical switch, for example, a planar optical switch. The switchablelight source can be a light manipulating device for switching light froma given input to any given output. Moreover, the switchable light sourcecan multicast an input light to several outputs all at the same time. Inone embodiment, switchable light source is an optical switch coupled toa light generator through one or more optical fiber (not shown). In aparticular embodiment, the light generator is coupled to one or more ofthe inputs of the switchable light source. By way of non-limitingexamples, the light generator can provide variable wavelengths of light.In one embodiment, the light generator is a broad-band source. Inanother embodiment, the light generator is a tunable source.

The switchable light source can include K (=1, 2, 3 . . . ) inputs and Noutput. In some embodiments, the number of outputs will be equal to thenumber of excitation waveguides in the sensing substrate of the system.In a particular embodiment, the interface between a light generatingsource and the switchable light source inputs includes optical fibers.The interface between the switchable light source outputs should match,in terms of pitch, the excitation waveguides in the sensing substrate toallow these two elements to butt-couple and transfer light from theswitchable light source to the excitation waveguides of the sensingsubstrate.

In one embodiment the optical switch includes individual switchingelements based on Mach Zehnder interferometers.

The switchable light source can include an array of light generators. Inone implementation, the light generators are light emitting diodes(LED). In another implementation the light generators are laser chips.Each individual light generator is separately controlled and can be turnon or off as desired. In one implementation the switchable light sourceincludes 10 or more light generators. In another implementation theswitchable light source includes 100 or more light generators. In yetanother implementation the switchable light source includes 1000 or morelight generators. In a particular implementation the switchable lightsource includes between 10 and 100 light generators.

The light generator array on the switchable light source can beintegrated on a single chip which includes an array of two or more lightgenerators and an array of two or more waveguides. In one implementationeach light generator is optically coupled to one waveguide and adaptedsuch that most of the light emitted by the light generator propagatesalong the waveguide. The waveguides can extend to the edge of the chipwhere they can be brought to couple the light propagating within them tothe sensing chip. In one implementation two light generators, eachoptionally emitting at a different wavelength can be coupled to a singlewaveguide. In another implementation more than two light generators,each optionally emitting at a different wavelength can be coupled to asingle waveguide.

The switchable light source can include in addition to a light generatorarray and waveguide array, light manipulating features such as filters,switches, modulators, splitters, combiners, mirrors and circulators.

The control of the switchable light source can be either integrated onthe same chip as the light generators and waveguides or alternativelycan be external to the chip. The switchable light source can have anelectrical interface to an external driver or external controller orlogic interface to an external control system. The control of theswitchable light source allows driving each light generator separately.It further allows also control of the other features present on theswitchable light source such as, for example, the modulators andswitches.

The switchable light source can couple to the sensing substrate inseveral different ways. In one implementation the coupling is done bybringing the ends of the waveguides on both chips (the switchable lightsource and the sensing substrate) in close proximity and allowing thelight to flow directly from one waveguide to the other. In anotherimplementation, a portion of the waveguides on both chips are aligned ontop of each other, parallel and in close proximity to each other, thuscoupling light from one waveguide to the other through the evanescentelectromagnetic field.

Additional elements useful in planar lightwave circuits, including butare not limited to couplers, filters, mirrors, circulators, splitters,modulators, switches and trenches are envisioned as part of the systemdescribed herein (not shown). Such elements when integrated into thesensing substrate or into the switchable light source substrate canserve to manipulate both incoming first light waves in the excitationwaveguides or outgoing second light waves in the collection waveguides.

FIG. 7 illustrates an exemplary detector 706 of the system of theinvention including elements 707 (shown as M-elements). In oneembodiment, as shown in FIG. 7, the detector 706 includes an array oflight sensitive elements 707, for example, in the form of aphotodetector array. In one embodiment, as shown in FIG. 1A, the numberof elements 116 matches the number of collection waveguides 110 in thesubstrate 104. Typically, the elements 116 are aligned with thecollection waveguides 110 and provided in a one to one ratio.

In one non-limiting example, the detector is a detector array having aspectral range of between 400 to 1000 nm, a photosensitivity (A/W)of >0.3, an active area per element of 0.005 mm², 128 elements, and apitch of <0.1 mm.

In one embodiment, the detector is a silicon photodiode (PN, PIN or APD)array. An example of a suitable detector array is the Texas AdvancedOptoelectronic Solutions (TAOS) 1×128 linear array (PN-TSL1210R).

It is envisioned that the element size and the pitch between theelements can equal that of the collection waveguides in the substrate.

A control system for managing the different steps of operating thescanning sensing system is envisioned. The control system can managesteps such as alignment of the switchable light source, sensingsubstrate and detector, in addition to switching the light output fromthe switchable light source, reading the detector array and reportingthe results detected.

In practicing the methods of the present invention, many conventionaltechniques in molecular biology are optionally utilized. Thesetechniques are well known and are explained in, for example, Ausubel etal. (Eds.) Current Protocols in Molecular Biology, Volumes I, II, andIII, (1997), Ausubel et al. (Eds.), Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, 5th Ed., John Wiley & Sons, Inc. (2002), Sambrook et al.,Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring HarborLaboratory Press (2000), and Innis et al. (Eds.) PCR Protocols: A Guideto Methods and Applications, Elsevier Science & Technology Books (1990),all of which are incorporated herein by reference.

Sample preparation suitable for use with the system and methodsdescribed herein can include any of a number of well know methods forcollection and analysis of biological and/or environmental samples. Inthe case of biological samples the sample can be, for example,manipulated, treated, or extracted to any desired level of purity for atarget of interest.

The sample can be bodily fluids suspected to contain a biologicallyactive analyte. Commonly employed bodily fluids include but are notlimited to blood, serum, saliva, urine, gastric and digestive fluid,tears, stool, semen, vaginal fluid, interstitial fluids derived fromtumorous tissue, and cerebrospinal fluid.

It is anticipated that the systems described herein can be used forscreening a large variety of samples. In the case where the investigatedsubject is a living creature, the sample may originate from body fluidsas discussed. Methods of obtaining samples include but are not limitedto cheek swabbing, nose swabbing, rectal swabbing, skin fat extractionor other collection strategies for obtaining a biological or chemicalsubstance. When the tested subject is a non-living or environmentalbody, the sample may originate from any substance in a solid phase,liquid phase or gaseous phase. The sample may be collected and placedonto the sensing substrate or the sensing substrate may be directlyexposed to the investigated sample source (e.g. water reservoir, freeair) and interact with it.

In some embodiments, the bodily fluids are used directly for detectingone or more biologically active analyte present therein with the subjectscanning sensing device without further processing. Where desired,however, the bodily fluids can be pre-treated before performing theanalysis with the subject scanning sensing devices. The choice ofpre-treatments will depend on the type of bodily fluid used and/or thenature of the biologically active analyte under investigation. Forinstance, where the biologically active analyte is present at low levelin a sample of bodily fluid, the sample can be concentrated via anyconventional means to enrich the biologically active analyte. Methods ofconcentrating a biologically active analyte include but are not limitedto drying, evaporation, centrifugation, sedimentation, precipitation,and amplification. Where the biologically active analyte is a nucleicacid, it can be extracted using various lytic enzymes or chemicalsolutions according to the procedures set forth in Sambrook et al.(“Molecular Cloning: A Laboratory Manual”), or using nucleic acidbinding resins following the accompanying instructions provided bymanufactures. Where the biologically active analyte is a moleculepresent on or within a cell, extraction can be performed using lysingagents including but not limited to denaturing detergent such as SDS ornon-denaturing detergent such as thesit (2-dodecoxyethanol), sodiumdeoxylate, Triton® X-100, and Tween® 20.

In some embodiments, pretreatment can include diluting and/or mixing thesample, and filtering the sample to remove, e.g., red blood cells from ablood sample.

Targets detectable using the scanning sensing system include but are notlimited to, a biologically active analyte including a nucleic acid, aprotein, an antigen, an antibody, a microorganism, a gas, a chemicalagent and a pollutant.

In one embodiment, the target is a nucleic acid that is DNA, forexample, cDNA. In a related embodiment, the DNA target is produced viaan amplification reaction, for example, by polymerase chain reaction(PCR). In another embodiment of the subject invention, the detectedbiologically active analyte is a protein representing a known bio-markerfor a disease or specific condition of the investigated organism. Inanother embodiment several different biologically active analytes can beproteins provided as a panel of bio-markers wherein relativeconcentrations of the bio-markers are indicative for a disease or othercondition of the investigated organism. In a further embodiment thetarget is a microorganism that is a pathogen. In another embodiment thetarget is a chemical agent, for example, a toxic chemical agent.

Where the target is a nucleic acid, it can be single-stranded,double-stranded, or higher order, and can be linear or circular.Exemplary single-stranded target nucleic acids include mRNA, rRNA, tRNA,hnRNA, ssRNA or ssDNA viral genomes, although these nucleic acids maycontain internally complementary sequences and significant secondarystructure. Exemplary double-stranded target nucleic acids includegenomic DNA, mitochondrial DNA, chloroplast DNA, dsRNA or dsDNA viralgenomes, plasmids, phage, and viroids. The target nucleic acid can beprepared synthetically or purified from a biological source. The targetnucleic acid may be purified to remove or diminish one or more undesiredcomponents of the sample or to concentrate the target nucleic acids.Conversely, where the target nucleic acid is too concentrated for theparticular assay, the target nucleic acid may be diluted.

Following sample collection and optional nucleic acid extraction, thenucleic acid portion of the sample comprising the target nucleic acidcan be subjected to one or more preparative reactions. These preparativereactions can include in vitro transcription (IVT), labeling,fragmentation, amplification and other reactions. MRNA can first betreated with reverse transcriptase and a primer to create cDNA prior todetection and/or amplification; this can be done in vitro with purifiedmRNA or in situ, e.g. in cells or tissues affixed to a slide. Nucleicacid amplification increases the copy number of sequences of interestsuch as the target nucleic acid. A variety of amplification methods aresuitable for use, including the polymerase chain reaction method (PCR),the ligase chain reaction (LCR), self sustained sequence replication(3SR), nucleic acid sequence-based amplification (NASBA), the use of QBeta replicase, reverse transcription, nick translation, and the like.

Where the target nucleic acid is single-stranded, the first cycle ofamplification forms a primer extension product complementary to thetarget nucleic acid. If the target nucleic acid is single stranded RNA,a polymerase with reverse transcriptase activity is used in the firstamplification to reverse transcribe the RNA to DNA, and additionalamplification cycles can be performed to copy the primer extensionproducts. The primers for a PCR must, of course, be designed tohybridize to regions in their corresponding template that will producean amplifiable segment; thus, each primer must hybridize so that its 3′nucleotide is paired to a nucleotide in its complementary templatestrand that is located 3′ from the 3′ nucleotide of the primer used toreplicate that complementary template strand in the PCR.

The target nucleic acid can be amplified by contacting one or morestrands of the target nucleic acid with a primer and a polymerase havingsuitable activity to extend the primer and copy the target nucleic acidto produce a full length complementary nucleic acid or a smaller portionthereof. Any enzyme having a polymerase activity that can copy thetarget nucleic acid can be used, including DNA polymerases, RNApolymerases, reverse transcriptases, enzymes having more than one typeof polymerase activity, and the enzyme can be thermolabile orthermostable. Mixtures of enzymes can also be used. Exemplary enzymesinclude: DNA polymerases such as DNA Polymerase I (“Pol I”), the Klenowfragment of Pol I, T4, T7, Sequenase® T7, Sequenase® Version 2.0 T7,Tub, Tag, Tth, Pfx, Pfu, Tsp, Tfl, Tli and Pyrococcus sp GB D DNApolymerases; RNA polymerases such as E. coli, SP6, T3 and T7 RNApolymerases; and reverse transcriptases such as AMV, M MuLV, MMLV, RNAseH′ MMLV (Superscript®), Superscript® II, ThermoScript®, HIV 1, and RAV2reverse transcriptases. All of these enzymes are commercially available.Exemplary polymerases with multiple specificities include RAV2 and Tli(exo) polymerases. Exemplary thermostable polymerases include Tub, Taq,Tth, Pfx, Pfu, Tsp, Tfl, Tli and Pyrococcus sp. GB D DNA polymerases.

Suitable reaction conditions are chosen to permit amplification of thetarget nucleic acid, including pH, buffer, ionic strength, presence andconcentration of one or more salts, presence and concentration ofreactants and cofactors such as nucleotides and magnesium and/or othermetal ions (e.g., manganese), optional cosolvents, temperature, thermalcycling profile for amplification schemes comprising a polymerase chainreaction, and may depend in part on the polymerase being used as well asthe nature of the sample. Cosolvents include formamide (typically atfrom about 2 to about 10%), glycerol (typically at from about 5 to about10%), and DMSO (typically at from about 0.9 to about 10%). Techniquesmay be used in the amplification scheme in order to minimize theproduction of false positives or artifacts produced duringamplification. These include “touchdown” PCR, hot start techniques, useof nested primers, or designing PCR primers so that they form stem-loopstructures in the event of primer-dimer formation and thus are notamplified. Techniques to accelerate PCR can be used, for example,centrifugal PCR, which allows for greater convection within the sample,and comprising infrared heating steps for rapid heating and cooling ofthe sample. One or more cycles of amplification can be performed. Anexcess of one primer can be used to produce an excess of one primerextension product during PCR; preferably, the primer extension productproduced in excess is the amplification product to be detected. Aplurality of different primers may be used to amplify different targetnucleic acids or different regions of a particular target nucleic acidwithin the sample.

Amplified target nucleic acids may be subjected to post amplificationtreatments. For example, in some cases, it may be desirable to fragmentthe target nucleic acid prior to hybridization in order to providesegments which are more readily accessible. Fragmentation of the nucleicacids can be carried out by any method producing fragments of a sizeuseful in the assay being performed; suitable physical, chemical andenzymatic methods are known in the art.

An amplification reaction can be performed under conditions which allowa nucleic acid associated with the optical sensing site to hybridize tothe amplification product during at least part of an amplificationcycle. When the assay is performed in this manner, real time detectionof this hybridization event can take place by monitoring for lightemission during amplification.

Real time PCR product analysis (and related real timereverse-transcription PCR) provides a well-known technique for real timePCR monitoring that has been used in a variety of contexts, which can beadapted for use with the methods described herein (see, Laurendeau etal. (1999) “TaqMan PCR-based gene dosage assay for predictive testing inindividuals from a cancer family with INK4 locus haploinsufficiency”Clin Chem 45(7):982-6; Bièche et al. (1999) “Quantitation of MYC geneexpression in sporadic breast tumors with a real-time reversetranscription-PCR assay” Cancer Res 59(12):2759-65; and Kreuzer et al.(1999) “LightCycler technology for the quantitation of bcr/abl fusiontranscripts” Cancer Res 59(13):3171-4, all of which are incorporated byreference). In addition, linear PCR and Linear-After-The Exponential(LATE)-PCR can be adapted for use with the methods described herein.

Immunoassays can be conducted on the scanning sensor system of theinvention, for example, at one or more optical sensing site of thesystem. Suitable immunoassay systems include but are not limited tocompetitive and non-competitive assay systems. Such assay systems aretypically used with techniques such as western blots, radioimmunoassays,EIA (enzyme immunoassay), ELISA (enzyme-linked immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays, protein A immunoassays, and cellularimmunostaining (fixed or native) assays to name but a few. Such assaysare routine and well known in the art (see, e.g., Ausubel et al.,supra). Immunoassay techniques particularly useful with the scanningsensor systems described herein include but are not limited to ELISA,“sandwich” immunoassays, and fluorescent immunoassays. Exemplaryimmunoassays are described briefly below (but are not intended by way oflimitation).

ELISAs generally involve preparing antigen, coating a well (e.g., anoptical sensing site of the scanning sensor system) with the antigen,adding the antibody of interest conjugated to a detectable compound suchas an enzymatic substrate (e.g., horseradish peroxidase or alkalinephosphatase) to the well and incubating for a period of time, anddetecting the presence of the antigen. In ELISAs the antibody ofinterest does not have to be conjugated to a detectable compound;instead, a second antibody (which recognizes the antibody of interest)conjugated to a detectable compound may be added to the well. Further,instead of coating the well with the antigen, the antibody may be coatedto the well. In this case, a second antibody conjugated to a detectablecompound may be added following the addition of the antigen of interestto the coated well. One of skill in the art would be knowledgeable as tothe parameters that can be modified to increase the signal detected aswell as other variations of ELISAs known in the art.

In one exemplary immunoassay, a sample contains an unknown amount ofbiologically active analyte to be measured, which may be, for example, aprotein. The analyte may also be termed an antigen. The sample may bespiked with a known or fixed amount of labeled analyte. The spikedsample is then incubated with an antibody that binds to the analyte, sothat the analyte in the sample and the labeled analyte added to thesample compete for binding to the available antibody binding sites. Moreor less of the labeled analyte will be able to bind to the antibodybinding sites, depending on the relative concentration of the unlabeledanalyte present in the sample. Accordingly, when the amount of labeledanalyte bound to the antibody is measured, it is inversely proportionalto the amount of unlabeled analyte in the sample. The amount of analytein the original sample may then be calculated based on the amount oflabeled analyte measured, using standard techniques in the art.

In one exemplary competitive immunoassay, an antibody that binds to abiologically active analyte may be coupled with or conjugated with aligand, wherein the ligand binds to an additional antibody added to thesample being tested. One example of such a ligand includes fluorescein.The additional antibody may be bound to a solid support (e.g., anoptical sensing site of the scanning sensor system). The additionalantibody binds to the ligand coupled with the antibody that binds inturn to the analyte or alternatively to the labeled analyte, forming amass complex which allows isolation and measurement of the signalgenerated by the label coupled with the labeled analyte.

In another type of exemplary competitive immunoassay, the biologicallyactive analyte to be measured may be bound to a solid support (e.g., anoptical sensing site of the scanning sensor system), and incubated withboth an antibody that binds to the analyte and a sample containing theanalyte to be measured. The antibody binds to either the analyte boundto the solid support or to the analyte in the sample, in relativeproportions depending on the concentration of the analyte in the sample.The antibody that binds to the analyte bound to the solid support isthen bound to another antibody, such as anti-mouse IgG, that is coupledwith a label. The amount of signal generated from the label is thendetected to measure the amount of antibody that bound to the analytebound to the solid support. Such a measurement will be inverselyproportional to the amount of analyte present in the sample. Such anassay may be used in the scanning sensor system of the presentinvention.

A wide diversity of labels are available in the art that can be employedfor conducting the subject assays. In some embodiments labels aredetectable by spectroscopic, photochemical, biochemical, immunochemical,or chemical means. For example, useful nucleic acid labels includefluorescent dyes, enzymes, biotin, dioxigenin, or haptens and proteinsfor which antisera or monoclonal antibodies are available. A widevariety of labels suitable for labeling biological components are knownand are reported extensively in both the scientific and patentliterature, and are generally applicable to the present invention forthe labeling of biological components. Suitable labels include enzymes,substrates, cofactors, inhibitors, fluorescent moieties,chemiluminescent moieties, or bioluminescent labels. Labeling agentsoptionally include, for example, monoclonal antibodies, polyclonalantibodies, proteins, or other polymers such as affinity matrices,carbohydrates or lipids. Detection proceeds by any of the methodsdescribed herein, for example, by detecting an optical signal in anoptical waveguide. A detectable moiety can be of any material having adetectable physical or chemical property. Such detectable labels havebeen well-developed in the field of gel electrophoresis, columnchromatography, solid substrates, spectroscopic techniques, and thelike, and in general, labels useful in such methods can be applied tothe present invention. Preferred labels include labels that produce anoptical signal. Thus, a label includes without limitation anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical, thermal, or chemical means.

In some embodiments the label is coupled directly or indirectly to amolecule to be detected such as a product, substrate, or enzyme,according to methods well known in the art. As indicated above, a widevariety of labels are used, with the choice of label depending on thesensitivity required, ease of conjugation of the compound, stabilityrequirements, available instrumentation, and disposal provisions. Nonradioactive labels are often attached by indirect means. Generally, aligand molecule is covalently bound to a polymer. The ligand then bindsto an anti-ligand molecule which is either inherently detectable orcovalently bound to a signal system, such as a detectable enzyme, afluorescent compound, or a chemiluminescent compound. A number ofligands and anti-ligands can be used. Where a ligand has a naturalanti-ligand, for example, biotin, thyroxine, and cortisol, it can beused in conjunction with labeled, anti-ligands. Alternatively, anyhaptenic or antigenic compound can be used in combination with anantibody.

In some embodiments the label can also be conjugated directly to signalgenerating compounds, for example, by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,and umbelliferone. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, such as luminol.

Methods of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence by, for example, ascanning sensor system as described herein. Similarly, enzymatic labelsare detected by providing appropriate substrates for the enzyme anddetecting the resulting reaction product (e.g., a reaction productcapable of producing a detectable optical signal).

In some embodiments the detectable signal may be provided byluminescence sources. “Luminescence” is the term commonly used to referto the emission of light from a substance for any reason other than arise in its temperature. In general, atoms or molecules emit photons ofelectromagnetic energy (e.g., light) when they move from an “excitedstate” to a lower energy state (usually the ground state); this processis often referred to as “radioactive decay”. There are many causes ofexcitation. If the exciting cause is a photon, the luminescence processis referred to as “photoluminescence”. If the exciting cause is anelectron, the luminescence process is referred to as“electroluminescence”. More specifically, electroluminescence resultsfrom the direct injection and removal of electrons to form anelectron-hole pair, and subsequent recombination of the electron-holepair to emit a photon. Luminescence which results from a chemicalreaction is usually referred to as “chemiluminescence”. Luminescenceproduced by a living organism is usually referred to as“bioluminescence”. If photoluminescence is the result of a spin allowedtransition (e.g., a single-singlet transition, triplet-triplettransition), the photoluminescence process is usually referred to as“fluorescence”. Typically, fluorescence emissions do not persist afterthe exciting cause is removed as a result of short-lived excited stateswhich may rapidly relax through such spin allowed transitions. Ifphotoluminescence is the result of a spin forbidden transition (e.g., atriplet-singlet transition), the photoluminescence process is usuallyreferred to as ”phosphorescence”. Typically, phosphorescence emissionspersist long after the exciting cause is removed as a result oflong-lived excited states which may relax only through suchspin-forbidden transitions. A “luminescent label” may have any one ofthe above-described properties.

Suitable chemiluminescent sources include a compound which becomeselectronically excited by a chemical reaction and may then emit lightwhich serves as the detectible signal or donates energy to a fluorescentacceptor. A diverse number of families of compounds have been found toprovide chemiluminescence under a variety or conditions. One family ofcompounds is 2,3-dihydro-1,4-phthalazinedione. A frequently usedcompound is luminol, which is a 5-amino compound. Other members of thefamily include the 5-amino-6,7,8-trimethoxy- and thedimethylamino[ca]benz analog. These compounds can be made to luminescewith alkaline hydrogen peroxide or calcium hypochlorite and base.Another family of compounds is the 2,4,5-triphenylimidazoles, withlophine as the common name for the parent product. Chemiluminescentanalogs include para-dimethylamino and -methoxy substituents.Chemiluminescence may also be obtained with oxalates, usually oxalylactive esters, for example, p-nitrophenyl and a peroxide such ashydrogen peroxide, under basic conditions. Other useful chemiluminescentcompounds that are also known include —N-alkyl acridinum esters anddioxetanes. Alternatively, luciferins may be used in conjunction withluciferase or lucigenins to provide bioluminescence.

In a separate embodiment, the present invention provides a method ofmonitoring one or more pharmacological parameter, for example,Pharmacodynamic (PD) and/or pharmacokinetic (PK) parameters, useful forassessing efficacy and/or toxicity of a therapeutic agent. The methodcomprises subjecting a sample of bodily fluid from a subjectadministered with the therapeutic agent to a scanning sensing device formonitoring the one or more pharmacological parameter, the scanningsensing device can be used as described herein to yield detectablesignals indicative of the values of the more than one pharmacologicalparameter from the sample; and detecting the detectable signal generatedfrom said sample of bodily fluid.

In one implementation the samples tested can include a large number of avariety of small molecules (e.g., screening libraries) which are ofinterest when investigating new drugs. Accordingly, the scanning sensingsystem described herein is useful for screening libraries of smallmolecules to investigate their ability to interact with certainbiologically active analytes may reveal potential new drugs. Furtherscreening of some or all small molecule candidates may reveal adversedrug effects and toxicity.

In one implementation the samples can include molecules which are testedfor toxicity.

In general in another aspect methods of using the scanning sensingsystem described herein are provided. In one embodiment, the switchablelight source, for example, an optical switch or an array of lightgenerators, couples light into one or more excitation waveguides at anygiven time. The light travels along the excitation waveguides, reachesthe optical sensing sites and interacts through the sensor, for example,an optical transducer. The samples are positioned at or near theexcitation waveguides. Next, the light leaving the sensor couples intothe collection waveguides and travels down the waveguide to its end atan edge of the substrate, for example, a chip facet. Light exiting thecollection waveguides is then detected by the different elements of thedetector, which can be a detector array.

In another embodiment, scanning sensing of a sample includes deliveringa sample suspected of containing a target to be detected to an opticalsensing site of the scanning sensor system. Delivering a sample to thesystem can include pipetting of a fluid to the optical sensing site.Other delivery means can include but are not limited to robotic fluiddelivery system or physically depositing a non-fluid or semi-fluidsample at the optical sensing site, either by hand or with the aid of atool or robot manipulation system. Next, a first light wave produced bythe switchable light source is provided to one or more of the pluralityof substantially parallel excitation waveguides in optical communicationwith the optical sensing site. The first light wave is transduced (e.g.,measurably changed) by the sensor associated with the optical sensingsite to form a second light wave carried in one or more of the pluralityof substantially parallel collection waveguides which are in opticalcommunication with the optical sensing site and cross the excitationwaveguides. Next a measurable change in the second light wave isdetected using the detector which is in optical communication with thecollection waveguides. Detection of measurable change in the secondlight waves indicates that the sensor has interacted with the target.

In a further embodiment, scanning sensing includes switching one or morelight wave from the switchable light source into the substrate toproduce the first light wave in one or more of the excitation waveguidesin a controlled and scanning manner.

In another embodiment, the switchable light source comprises an opticalswitch for controlling switching of one or more input light wave. Theoptical switch can multicast light to a plurality of outputs. Theplurality of light waves can be coupled into the sensing substrate tocontrollably produce the first light wave in one or more of theexcitation waveguides.

In one embodiment, a single excitation waveguide is provided with afirst light wave and simultaneous detection of second light waves ateach collection waveguide is achieved using a detector that is aphotodetector array. By switching light between excitation waveguides,each individual excitation waveguide can be individually addressed witha first light wave. The order of addressing the excitation waveguidescan be sequential, staggered, random or in any order desired. Rapidscanning of the entire two-dimensional array of optical sensing sitescan be achieved with the aid of the photodetector array since any secondlight wave associated with each collection waveguide can besimultaneously detected. For example, where the two-dimensional array isconfigured as an array of 128 excitation waveguides and 128 collectionwaveguides, then it would be possible to simultaneously detect secondlight waves (if any) generated from 128 optical sensor sites afterproviding a single first lightwave in a first excitation waveguide.Thus, 128 optical sensing sites can be interrogated for presence orabsence of target simultaneously. Next, a second excitation waveguidecan be excited thereby triggering the interrogation of a second set of128 optical sensing sites. The process can rapidly be repeated untilevery excitation waveguide has been excited and the entire array ofoptical sensing sites have been interrogated.

In various embodiments the method of using the scanning sensing systeminvolves the detection of a substance, including but not limited to abiologically active analyte including a nucleic acid, a protein, anantigen, an antibody, a panel of proteins, a microorganism, a gas, achemical agent and a pollutant. In a particular embodiment, a singlenucleotide polymorphism (SNP) is detected in the target. In oneembodiment expression of a gene is detected upon detection of thetarget.

In one embodiment immunoassays can be used with the present method ofusing the scanning sensing system. The sensor of the method of using thescanning sensing system of the invention can be adapted to support animmunoassay, for example, by including one or more immunoassay reagentat or within the sensor. In this embodiment an interaction between thesensor and a sample being tested for a biologically active analyte caninclude an immunoassay conducted at the sensor. As such, the sensorinteracting with the biologically active analyte can include an outcomeof an immunoassay. In this manner, presence or absence of the analytecan be determined. Additionally the amount of analyte can be quantified.In one embodiment the immunoassay supported is a fluorescent assay. Itis envisioned that the immunoassay can be a competitive ornon-competitive immunoassay. In one embodiment the immunoassay supportedin an ELISA.

It is envisioned that a variety of instrumentation relating tobiological or environmental sample preparation, handling and analysiscan be used in conjunction with the system and methods described herein.Examples of such instrumentation include but are not limited to a cellsorter, a DNA amplification thermal cycler, or a chromatography machine(e.g., GC or HPLC). Such instrumentation is well known to those skilledin the art. It is envisioned that a robotic interface could be usedbetween the scanning sensing system of the present invention and variousinstrumentation relating to biological or environmental samplepreparation, handling and analysis.

The system and methods described herein may be used in a range ofapplications including biomedical and genetic research as well asclinical diagnostics. Arrays of polymers such as nucleic acids may bescreened for specific binding to a target, such as a complementarynucleotide, for example, in screening studies for determination ofbinding affinity and in diagnostic assays. In one embodiment, sequencingof polynucleotides can be conducted, as disclosed in U.S. Pat. No.5,547,839. The nucleic acid arrays may be used in many otherapplications including detection of genetic diseases such as cysticfibrosis or diagnosis of HIV, as disclosed in U.S. Pat. No. 6,027,880and U.S. Pat. No. 5,861,242. Genetic mutations may be detected bysequencing by hydridization. In one embodiment, genetic markers may besequenced and mapped using Type-IIs restriction endonucleases asdisclosed in U.S. Pat. No. 5,710,000.

Other applications include chip based genotyping, species identificationand phenotypic characterization, as described in U.S. Pat. No.6,228,575. Still other applications including diagnosing a cancerouscondition or diagnosing viral, bacterial, and other pathological ornonpathological infections, are described in U.S. Pat. No. 5,800,992. Afurther application includes chip based single nucleotide polymorphism(SNP) detection as described in U.S. Pat. No. 6,361,947.

Gene expression may be monitored by hybridization of large numbers ofmRNAs in parallel using high density arrays of nucleic acids in cells,such as in microorganisms such as yeast, as described in Lockhart etal., Nature Biotechnology, 14:1675-1680 (1996). Bacterial transcriptimaging by hybridization of total RNA to nucleic acid arrays may beconducted as described in Saizieu et al., Nature Biotechnology, 16:45-48(1998). Accessing genetic information using high density DNA arrays isfurther described in Chee, Science 274:610-614 (1996).

A non-limiting list of potential application suitable for sensing usingthe systems and methods described herein includes: pathogens detectionand classification; chemical/biological warfare real-time detection;chemical concentration control; dangerous substance (e.g., gas, liquid)detection and alarm; sugar and insulin levels detection in diabeticpatients; pregnancy testing; detection of viral and bacterial infectiousdiseases (e.g. AIDS, Bird Flu, SARS, West Nile virus); environmentalpollution monitoring (e.g., water, air); and quality control in foodprocessing.

The working system described here can also be a sub-system within a muchlarger bio-analysis system. The bio-analysis system could include allthe aspects of sample preparation prior to the optical scanning, thepost processing of data collected in the optical scanning phase andfinally decision making based on these results. Sample preparation mayinclude steps such as: extraction of the sample from the tested subject(human, animal, plant environment etc.); separation of different partsof the sample to achieve higher concentration and purity of themolecules under investigation; sample amplification (e.g. through PCR);attachment of fluorescence tags or markers to different parts of thesample; and spotting of the sample into the sensing chip. The postprocessing of the collected data may include: normalization; backgroundand noise reduction; and statistical analysis such as averaging overrepeated tests or correlation between different tests. The decisionmaking may include: testing against a predefined set of rules andcomparison to information stored in external data-bases.

The applications and uses of the scanning sensing systems describedherein can produce one or more result useful to diagnose a disease stateof an individual, for example, a patient. In one embodiment, a method ofdiagnosing a disease comprises reviewing or analyzing data relating tothe presence and/or the concentration level of a target in a sample. Aconclusion based review or analysis of the data can be provided to apatient, a health care provider or a health care manager. In oneembodiment the conclusion is based on the review or analysis of dataregarding a disease diagnosis. It is envisioned that in anotherembodiment that providing a conclusion to a patient, a health careprovider or a health care manager includes transmission of the data overa network.

Accordingly, business systems and methods using the scanning sensingsystems and methods described herein are provided.

One aspect of the invention is a business method comprising screeningpatient test samples for the presence or absence of a biologicallyactive analyte to produce data regarding the analyte, collecting theanalyte data, providing the analyte data to a patient, a health careprovider or a health care manager for making a conclusion based onreview or analysis of the data regarding a disease diagnosis. In oneembodiment the conclusion is provided to a patient, a health careprovider or a health care manager includes transmission of the data overa network.

Accordingly FIG. 8 is a block diagram showing a representative examplelogic device through which reviewing or analyzing data relating to thepresent invention can be achieved. Such data can be in relation to adisease, disorder or condition in an individual. FIG. 8 shows a computersystem (or digital device) 800 connected to an apparatus 820 for usewith the scanning sensing system 824 to, for example, produce a result.The computer system 800 may be understood as a logical apparatus thatcan read instructions from media 811 and/or network port 805, which canoptionally be connected to server 809 having fixed media 812. The systemshown in FIG. 8 includes CPU 801, disk drives 803, optional inputdevices such as keyboard 815 and/or mouse 816 and optional monitor 807.Data communication can be achieved through the indicated communicationmedium to a server 809 at a local or a remote location. Thecommunication medium can include any means of transmitting and/orreceiving data. For example, the communication medium can be a networkconnection, a wireless connection or an internet connection. Such aconnection can provide for communication over the World Wide Web. It isenvisioned that data relating to the present invention can betransmitted over such networks or connections for reception and/orreview by a party 822. The receiving party 822 can be but is not limitedto a patient, a health care provider or a health care manager.

In one embodiment, a computer-readable medium includes a medium suitablefor transmission of a result of an analysis of an environmental orbiological sample. The medium can include a result regarding a diseasecondition or state of a subject, wherein such a result is derived usingthe methods described herein.

Kits comprising reagents useful for performing the methods describedherein are also provided.

In some embodiments, a kit comprises scanning sensing system asdescribed herein and reagents for detecting a target in the sample. Thekit may optionally contain one or more of the following: one or morefluorescent or luminescent molecular tag, and one or more biologicallyactive analyte including a nucleic acid, protein, microorganism orchemical agent.

The components of a kit can be retained by a housing. Instructions forusing the kit to perform a described method can be provided with thehousing, and can be provided in any fixed medium. The instructions maybe located inside the housing or outside the housing, and may be printedon the interior or exterior of any surface forming the housing thatrenders the instructions legible. A kit may be in multiplex form fordetection of one or more different target biologically active analyteincluding nucleic acid, protein, microorganism, gas, chemical agent orpollutant.

As described herein and shown in an illustrative example in FIG. 9, incertain embodiments a kit 903 can include a housing or container 902 forhousing various components. As shown in FIG. 9, and described herein,the kit 903 can optionally include instructions 901 and reagents 905,for example, DNA hybridization or immunoassay reagents. Otherembodiments of the kit 903 are envisioned wherein the components includevarious additional features described herein.

In one embodiment, a kit for assaying a sample for a target includes ascanning sensor system including a switchable light source, a detector,and a substrate. The substrate includes a plurality of substantiallyparallel excitation waveguides and a plurality of substantially parallelcollection waveguides. The excitation waveguides and collectionwaveguides of the substrate cross or intersect to form intersectionregions and a two-dimensional array. The system further includes aplurality of optical sensing sites including sensors. The opticalsensing sites are in optical communication with one or more excitationwaveguides and one or more collection waveguides. The kit furtherincludes packaging and instructions for use of the system.

In one embodiment, the kit includes a scanning sensor system that is aplanar lightwave circuit. In another embodiment, the crossing of theexcitation waveguides and collection waveguides is substantiallyperpendicular.

In general, in another aspect methods of manufacturing a scanningsensing system for assaying a sample for a target are provided. In oneembodiment the system is a planar lightwave circuit (PLC).

The starting material or substrate for manufacturing PLC devices is awafer usually made of Silicon (Si) or Silica (SiO2). The most commonwafer diameters in use are 4″, 6″ and 8″. The manufacturing process forPLC devices involves two basic processes namely, deposition and etching.A short description of each of them is given below.

In certain embodiments the methods of manufacturing the systemsdescribed herein can include, but are not limited to laser writing, UVwriting and photonic band-gap waveguide methods. The manufacturingprocess in some embodiments includes one or more steps of deposition,masking and etching.

Deposition

In the deposition step a layer of well defined material having wellcontrolled thickness is deposited across the entire wafer. The mostcommon material used for waveguide layer deposition is Silica (SiO2)also known as glass. The optical properties of the Silica (mainly itsrefractive index) is controlled by the amount of doping (Ge, P, and Betc.) introduced during the deposition. Other materials such as silicon,glass, epoxy, lithium niobate, indium phosphide and SiON (SiliconOxyNitride) and its derivatives are also used. For the cladding layer,materials can include but are not limited to silicon, silica (SiO2),glass, epoxy, lithium niobate and indium phosphide

The deposition step is done using several technologies such as PECVD(Plasma-Enhanced Chemical Vapor Deposition), LPCVD (Low Pressure CVD),APCVD (Atmospheric pressure CVD), FHD (Flame Hydrolysis Deposition) andothers well known in the art.

FIG. 10A illustrates an exemplary substrate 1004 as a schematicstructure created after two consecutive deposition steps of a cladding1021 layer and a core 1023 layer over a silicon 1020 layer, which can bea wafer. As mentioned above, these two layers differ in the refractionindex which is achieved by using different levels of doping. Typicalthicknesses for the different layers are: Cladding up to about 20 μm andcore up to 6 μm. The thickness of the silicon 1020 wafer can range fromabout 0.5 mm to 1 mm.

Masking

Following the deposition and before the etching step, the desiredtwo-dimensional structure of the PLC device is transferred to thedeposited wafer by masking the areas not to be etched away. The maskingis done in several steps involving covering the wafer with lightsensitive material, exposing it to light through lithographic masks andremoving the exposed material leaving in place the mask. The result ofsuch steps is shown in FIG. 10B where a mask 1025 is shown on top of thecore 1023 layer of the substrate 1004.

Etching

In the etching step, material at the un-masked areas is removed from thetop core 1023 layer of the substrate (see FIG. 10C). The etching rate isa known parameter, therefore the etching depth can be controlled bytime. The two most common techniques for etching are wet-etching andReactive-Ion-Etching (RIO). FIG. 10C shows the results of the etchingstep which results in a waveguide 1027.

After the etching step, an over-cladding or top cladding 1029 layer iscreated using a deposition step similar to the one described above. Theresults are shown in FIG. 10D. As shown in FIG. 10D, the resultingwaveguide 1027 can be surrounded by a top cladding 1029 and a cladding1021 over a silicon 1020 layer.

The above steps can be repeated to create several waveguide layers oneon top of the other (see for example, FIG. 3). In this case, aplanarization step is required between one waveguide layer and theother. This is done using a technique known as Chemical MechanicalPlanarization (CMP).

When the wafer processing is completed, it can be diced into theindividual chips. An exemplary simplified flow-chart of themanufacturing process is shown in FIG. 11.

EXAMPLES Prophetic Example 1

Quantitative real-time PCR detection of transcripts, for example,bcr/abl fusion transcripts, can be achieved using a scanning sensingsystem such as the example illustrated in FIG. 1A using a PCR assayprotocol modified from that described by Kreuzer et al. (supra). Thesystem includes a substrate chip with excitation waveguides, collectionwaveguides and intersection regions. The intersection regions where theexcitation waveguides and collection waveguides cross include opticalsensing sites with sensing wells for conducting quantitative real-timePCR. The scanning sensing system further includes a dynamic switchablelight source coupled to the substrate and the excitation waveguides. Thesystem further includes an optical detector.

Samples suspected of containing RNA transcripts of interest (e.g., bloodfrom a subject) are treated to obtain a source of total RNA which issubsequently reverse transcribed using reverse transcriptase into cDNAusing techniques well known in the art. cDNA samples are delivered tosensing sites on the substrate of the system. As desired, suitablecontrols and different dilutions of a particular cDNA sample can bedelivered to different sensing wells.

Reagents for real-time reverse transcriptase PCR are provided at thesensing sites. PCR reactions are conducted using primers/probes specificfor one or more bcr/abl breakpoint cluster region. Such primers/probesare well known in the art (e.g., see Kreuzer et al. supra) and can belabeled with 6-carboxy-fluorescein phosphoramidite at the 5′ end, and asa quencher, 5-carboxy-tetramethyl-rhodamine can be incorporated furtheralong the primers/probes sequence. As the probe is hydrolyzed throughthe 5′-nucelase activity of Taq DNA polymerase, unquenched fluorescencefrom the fluorescein (reporter) dye can be induced. Phosphate groups areattached to primers/probes 3′ end to prevent probe extension. A 10-μlPCR reaction mix contained 1 μl of 10× PCR buffer, 4.5 mM MgCl2, 0.8 mMdNTP, 0.5 μM each primer, 1 μM probe, 0.2 units of a temperature-releaseTaq DNA polymerase (Platinum® Pfx DNA Polymerase; Invitrogen, Corp.),and 20 ng of sample cDNA. PCR amplification is started with a 5-mindenaturation step at 94° C., followed by 45 cycles of denaturation at94° C. for 30 s and annealing/extension at 65° C. for 60 s.

While PCR amplification proceeds excitation light at a wavelength ofabout 472 nm is generated by the switchable light source and is directedto each sensing well by the excitation waveguides. If the cDNA samplesincluded a transcript for bcr/abl fusion transcripts, annealing ofprimers/probes to the cDNA should occur. Subsequent hydrolysis of theprobe by polymerase and unquenching of the fluorescein reporter resultsin fluorescence in the sensing well. As the number of cycles increases,the amount of unquenched fluorescein increases in relation to the amountof bcr/abl fusion transcript cDNA in the reaction.

By way of the collection waveguides fluorescence in the sensing wells isdetectable by the optical detector of the system. Thus, detection ofbcr/abl fusion transcripts can be measured in real-time and based onappropriate controls and analysis the amount of bcr/abl fusiontranscripts in a sample can be quantified.

Prophetic Example 2

Fluorescent immunoassay-based detection of HIV+ status of multiplesubjects can be achieved using a scanning sensing system such as theexample illustrated in FIG. 1A. The system includes a substrate chipwith excitation waveguides, collection waveguides and intersectionregions. The intersection regions where the excitation waveguides andcollection waveguides cross include optical sensing sites with sensingwells for conducting fluorescent immunoassays. The scanning sensingsystem further includes a dynamic switchable light source coupled to thesubstrate and the excitation waveguides. The system further includes anoptical detector.

A partially purified antigen, for example, inactivated HIV protein p29antigen, is pre-coated onto the sensing wells of the optical sensingsites. Next, a number subject serums which may contain antibodies to HIVp29 are delivered to separate sensing sites. If a subject is HIV+, thentheir serum may contain antibodies to HIV protein p29, and thoseantibodies will bind to the HIV p29 antigens on the sensing sites. Aftera washing step, anti-human immunoglobulin coupled to a fluorescent dye(fluorescein) is added to the sensing sites. This secondary antibody,binds to human antibodies in the sensing sites (i.e., the anti-p29antibodies). Next an excitation light at a wavelength of about 472 nm isgenerated by the switchable light source and is directed to each sensingwell by the excitation waveguides. If the secondary antibody is presentthe coupled fluorescein will fluoresce in the presence of the excitationlight in the well.

By way of the collection waveguides fluorescence in the sensing wells isdetectable by the optical detector of the system. Signal received by theoptical detectors can be interpreted to determine if a given subject hadantibodies to HIV p29. Suitable controls can be used to validate resultsof the assay. Thus, the presence or absence of HIV p29 can be measuredin a sample and HIV+ or HIV(−) status of multiple subjects can bedetermined.

Prophetic Example 3

A two-site sandwich immunoassay can be employed in assays using thescanning sensing system of the invention (e.g., the system asillustrated in FIG. 1A). Antibodies fulfill two different roles in suchassays; an immobilized antibody captures the analyte, while a soluble,fluorescently labeled antibody detects or “traces” analyte binding. Toprevent competitive binding, capture and tracer antibodies must bind todifferent sites on the analyte molecule. For large analytes withrepetitive epitopes (e.g., viruses and bacteria), a single antibody(specific for the repetitive epitope) can usually be employed in bothcapture and tracer roles. Smaller analytes (e.g. proteins andpolysaccharides) expressing multiple, unique epitopes require twodifferent antibodies, each specific for a unique epitope.

Three two-site sandwich immunoassay tests are envisioned: 1) serialtesting of optical sensing sites; 2) low complexity parallel testing ofoptical sensing sites; and 3) sensitivity testing. In the first ofthese, a small volume (1-5 μL) of sample (containing analyte and tracerantibody) is spotted directly at a optical sensing site containingcapture antibody using a microliter pipette. Binding kinetics at thesite are monitored over a 5-min period at room temperature. Translationof optical detection to excitation and collection waveguides inconnection with a different optical sensing site is effected and theassay is repeated at the new site. It is envisioned that at least 10optical sensing sites can be tested using this serial procedure. Suchtests can demonstrate sensitivity and intra-assay precision of thesystem.

In the second form of testing using a substrate of the system thatincludes a 10×10 array of excitation waveguides and collectionwaveguides (see e.g., FIG. 1A), a single excitation waveguide of thesubstrate is excited, while output signals are monitored from all 10collection waveguides using either an optical switch or a linear arrayof photodiodes. Equal volumes (e.g., 50 μL) of sample containing analyteand tracer antibody (e.g., 10 nM, final concentration) can be pre-mixedand then injected into a sample well of the optical sensing site thatcontains capture antibody. Binding kinetics are monitored simultaneouslyin 10 optical sensing sites over a 5-min period at room temperature.This form of testing can demonstrate the parallel assay capabilities ofthe system, as well as providing more detailed information aboutintra-assay precision.

In the third form of testing the sensitivity, precision and linearity ofthe device can be demonstrated by constructing a standard curve ofaverage reaction rate versus analyte concentration. Device configurationis the same as described above for the second form of testing (i.e., 10simultaneous assays). Analyte concentration is varied over at least a100-fold range, e.g. 10 pM to 1 nM, though the exact range can beadjusted depending on the clinical concentration range of the analytebeing examined. A separate substrate chip is used for each concentrationto be tested. Six to eight concentrations are examined. Resultingstandard curves are typically linear at low concentration, but saturateat higher concentrations.

The Herron lab has developed immunoassays for many different analytesincluding human cardiac troponin I (cTnI), chorionic gonadotrophin(hCG), creatine phosphokinase isoform MB (CKMB), myoglobin, ovalbumin(used by the military as a “simulant” for toxins such as ricin and SEB),ricin, Staphylococcal enterotoxin B (SEB). (see: Herron, J. N., H.-K.Wang, V. Janatová, J. D. Durtschi, K. D. Caldwell, D. A. Christensen,I.-N. Chang and S.-C. Huang (2003). Orientation and Activity ofImmobilized Antibodies. In: Biopolymers at Interfaces, 2nd Edition (M.Malmsten, ed.), Surfactant Science Series, Vol. 110, Marcel Dekker, NewYork, pp. 115-163; and Herron, J. N., H.-K. Wang, V. Janatová, J. D.Durtschi, K. D. Caldwell, D. A. Christensen, Durtschi, E. M. Simon, M.E. Astill, R. S. Smith and D. A. Christensen (2005). Planar WaveguideBiosensors for Point-Of-Care Clinical and Molecular Diagnostics. In:Fluorescence Sensors and Biosensors (R. B. Thompson, Ed.). CRC PressTaylor & Francis Group, Boca Raton, Fla. pp. 283-332)

The ovalbumin assay of Herron can be used in the first and secondimmunoassays described above. Advantageously, reagents for this assayare relatively inexpensive and no special handling is required.Detection requirements for cTnI and SEB are the most stringent, and thusimmunoassays specific for these analytes are useful for the sensitivitytesting immunoassays. However, since the CDC, NIH, and USDA all list SEBas a select agent requiring special handling it may be preferable to usecTnI in sensitivity testing. cTnI can be paired with two other cardiacmarkers (CKMB and myoglobin) for simultaneous immunoassay sensitivitytesting.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A scanning sensor system for detecting a biologically active analytecomprising: a switchable light source; a detector; a substrate coupledto and in optical communication with the switchable light source and thedetector, wherein the substrate comprises a plurality of substantiallyparallel excitation waveguides, and a plurality of substantiallyparallel collection waveguides, the excitation waveguides and collectionwaveguides crossing to form a two-dimensional array of intersectionregions where an excitation waveguide and a collection waveguide crossand provide optical communication with the intersection region at eachcrossing; and a plurality of optical sensing sites each in opticalcommunication with an intersection region.
 2. The system of claim 1,wherein the system is substantially planar.
 3. The system of claim 1,wherein the system comprises a planar lightwave circuit.
 4. The systemof claim 1, wherein the switchable light source is coupled to and incommunication with one or more of the excitation waveguides at a firstedge of the substrate and the detector is coupled to and incommunication with one or more of the collection waveguides at a secondedge of the substrate.
 5. The system of claim 1, wherein the opticalsensing sites comprise a sensor comprising a biologically active analytein a sample, and wherein a measurable change in the first light waveresults when the sensor discriminates or interacts with the biologicallyactive analyte.
 6. The system of claim 5 wherein a first light wavegenerated by the switchable light source in an excitation waveguide istransduced by a sensor of an optical sensing site in opticalcommunication with the excitation wave guide resulting in a second lightwave in a collection waveguide, the second light wave being detectableby the detector.
 7. The system of claim 1, wherein the sensor is adaptedto support an immunoassay.
 8. The system of claim 7, wherein theimmunoassay supported is an enzyme-linked immunosorbent assay (ELISA).9. The system of claim 7, wherein the immunoassay supported is afluorescent immunoassay.
 10. The system of claim 1, wherein the sensoris selected from the group consisting of a fluorescence well, anabsorption cell, an interferometric sensor, a diffractive sensor andsurface plasmon resonance sensor.
 11. The system of claim 1, wherein thebiologically active analyte is selected from the group consisting of anucleic acid, a protein, an antigen, an antibody, a microorganism, agas, a chemical agent and a pollutant.
 12. The system of claim 11,wherein the nucleic acid is produced via an amplification reaction. 13.The system of claim 1, wherein the excitation waveguides are single-modeand the collection waveguides are multi-mode.
 14. The system of claim 1,wherein the excitation waveguides support single-mode in a firstvertical dimension and multi-mode in a second lateral dimension andwherein the collection waveguides are multi-mode.
 15. The system ofclaim 1, wherein the excitation waveguides and the collection waveguidesare multi-mode.
 16. The system of claim 1, wherein the excitationwaveguides and the collection waveguides are single-mode.
 17. The systemof claim 1, wherein the excitation waveguide comprises a plurality ofbranches for drawing a fraction of the light from a first light wavetraveling in the excitation waveguide.
 18. The system of claim 17,wherein the excitation waveguide branches are in optical communicationwith the excitation waveguide.
 19. The system of claim 1, wherein thecollection waveguide comprises a plurality of funnels for collectinglight from the sensing sites and coupling it to the collectionwaveguide.
 20. The system of claim 1, wherein the optical sensing sitescomprise wells.
 21. The system of claim 1, wherein the optical sensingsites comprise the surface of the substrate above the intersectionregion of the excitation waveguides and the collection waveguides. 22.The system of claim 1, wherein the optical sensing sites comprisebiochemical interaction sites.
 23. The system of claim 1, wherein theoptical sensing sites comprise optical transducers.
 24. The system ofclaim 23, wherein the optical transducers comprise fluorescence wellscomprising fluorescent or luminescent compounds, wherein light wavesguided by the excitation waveguides excite the fluorescent orluminescent compound in the wells in the presence of a biologicallyactive analyte, and the collection waveguides collect and guide lightemitted from the wells to the detector.
 25. The system of claim 1,wherein the switchable light source comprises a dynamic light source.26. The system of claim 1, wherein the switchable light source comprisesa chip containing an array of light generators coupled to an array ofwaveguides.
 27. The system of claim 1, wherein the switchable lightsource is an optical switch comprising a light generator coupled to oneor more input of the optical switch.
 28. The system of claim 27, whereinthe optical switch further comprises a branched architecture.
 29. Thesystem of claim 28, wherein the optical switch further comprises one ormore inputs and multiple outputs.
 30. The system of claim 29, whereinthe optical switch further comprises greater than about 10 outputs. 31.The system of claim 29, wherein the optical switch further comprisesgreater than about 100 outputs.
 32. The system of claim 29, wherein theoptical switch further comprises greater than about 1,000 outputs. 33.The system of claim 29, wherein the optical switch further comprisessubstantially between 50 and 500 outputs.
 34. The system of claim 1,wherein the switchable light source is butt-coupled to the substrate.35. The system of claim 1, wherein the switchable light source comprisesone or more waveguide and is evanescently coupled to the substratethrough a proximate arrangement of the one or more switchable lightsource waveguide and one or more excitation waveguide of the substrate.36. The system of claim 27, wherein the light generator providesvariable wavelengths of light.
 37. The system of claim 27, wherein thelight generator is selected from the group consisting of a broad-bandsource, a source with one or more discrete spectral lines and a tunablesource.
 38. The system of claim 1, wherein the detector is aphotodetector array.
 39. The system of claim 1, wherein the detector isa plurality of detectors.
 40. The system of claim 39, wherein two ormore detectors are coupled to and in optical communication with one ormore of the collection waveguides or the excitation waveguides at one ormore edges of the substrate.
 41. The system of claim 1, wherein thenumber of intersection regions is greater than
 10. 42. The system ofclaim 1, wherein the density of intersection regions is greater than 100per cm².
 43. The system of claim 1, wherein the density of intersectionregions is greater than 2,000 per cm².
 44. The system of claim 1,wherein the system further comprises a thermal transfer element inthermal communication with the substrate.
 45. The system of claim 44,wherein the thermal transfer element is a thermoelectric cooler.
 46. Thesystem of claim 1, wherein each optical sensing site comprises a thermaltransfer element in thermal communication with the optical sensing site.47. The system of claim 46, wherein the thermal transfer elementcomprises a thin-film heater.
 48. The system of claim 46, wherein eachoptical sensing site further comprises a thermistor in thermalcommunication with the optical sensing site.
 49. The system of claim 1,wherein the substrate further comprises one or more microchannel and oneor more reservoirs in fluid communication with one or more opticalsensing site.
 50. The system of claim 1, wherein the system furthercomprises a fluidics layer coupled to the substrate and comprising oneor more microchannel and one or more reservoirs in fluid communicationwith one or more optical sensing site.
 51. A scanning sensing methodcomprising: delivering a sample suspected of containing a biologicallyactive analyte to be detected to an optical sensing site of a scanningsensor system; providing a first light wave using a switchable lightsource to one or more of a plurality of substantially parallelexcitation waveguides in optical communication with the optical sensingsite, wherein the first light wave is transducable by a sensorassociated with the optical sensing site to a second light wave carriedin one or more of a plurality of substantially parallel collectionwaveguides in optical communication with the optical sensing site andcrossing the excitation waveguides; and detecting a measurable change inthe second light wave using a detector in optical communication with thecollection waveguides, wherein a measurable change in the first lightwaves occurs when the sensor interacts with the biologically activeanalyte.
 52. The method of claim 51, wherein scanning sensing furthercomprises switching one or more input light wave from the switchablelight source into the substrate to produce the first light wave in oneor more of the excitation waveguides.
 53. The method of claim 51,wherein the switchable light source comprises an optical switch forcontrolled switching of one or more input light wave, the optical switchcan multicast light to a plurality of outputs and into the substrate tocontrollably produce the first light wave in one or more of theexcitation waveguides.
 54. The method of claim 51, wherein theswitchable light source comprises an array of individually controlledlight generators for controlled switching of one or more input lightwave, to controllably produce the first light wave in one or more of theexcitation waveguides.
 55. The method of claim 51, further comprisingsimultaneously detecting the second light wave with the detector at theend of each collection waveguide wherein the detector comprises aphotodetector array.
 56. The method of claim 51, wherein, a portion ofthe sensing sites comprise reference sample material for calibrationand/or normalization.
 57. The method of claim 51, wherein thebiologically active analyte is selected from the group consisting of anucleic acid, a protein, an antigen, an antibody, a microorganism, agas, a chemical agent and a pollutant.
 58. The method of claim 51,wherein the biologically active analyte is a protein.
 59. The method ofclaim 51, wherein a SNP is detected in the biologically active analyte.60. The method of claim 51, wherein expression of a gene is detectedupon detection of the biologically active analyte.
 61. The method ofclaim 51, wherein the sensor is adapted to support an immunoassay andwherein the sensor interacting with the biologically active analytecomprises an outcome of an immunoassay.
 62. The method of claim 61,wherein the immunoassay supported is an enzyme-linked immunosorbentassay (ELISA).
 63. The method of claim 61, wherein the immunoassaysupported is a fluorescent immunoassay.
 64. The method of claim 5 1,wherein detecting a measurable change in the second lightwave provides adiagnostic result.
 65. The method of claim 51, further comprisingconducting a real-time PCR reaction at the optical sensing site.
 66. Akit for assaying a sample for a biologically active analyte comprising:a scanning sensor system comprising a switchable light source, adetector, and a substrate coupled to and in optical communication withthe switchable light source and the detector, wherein the substratecomprises a plurality of substantially parallel excitation waveguides,and a plurality of substantially parallel collection waveguides, theexcitation waveguides and collection waveguides crossing to form atwo-dimensional array of intersection regions where an excitationwaveguide and a collection waveguide cross and provide opticalcommunication with the intersection regions at each crossing, and aplurality of optical sensing sites each in optical communication with anintersection region; packaging; and instructions for use of the system.67. The kit of claim 66, wherein the system comprises a planar lightwavecircuit.
 68. The kit of claim 66, wherein the crossing of the excitationwaveguides and collection waveguides is substantially perpendicular. 69.The kit of claim 66, wherein the optical sensing sites comprise a sensoradapted to support an immunoassay, and wherein the kit further comprisesone or more immunoassay reagents.